MSc Ralf Kuja-Halkola
email: ralf.kuja-halkola@ki.se.

Anastasia Nyman B D'onofrio

Niklas Iliadou

Paul Langstrom

Lichtenstein

Brian M D'onofrio

Anastasia N Iliadou

Niklas Långström

Paul Lichtenstein

Smoking during pregnancy and offspring stress coping 1 Prenatal smoking exposure and offspring stress coping in late adolescence: No causal link

Keywords: Causality, Smoking, Risk Factors, Pregnancy, Prenatal Exposure Delayed Effects, Stress, Psychological Smoking during pregnancy, adolescent stress coping, children-of-sibling model, intergenerational association

Introduction

Maternal smoking during pregnancy (SDP) has been linked to several negative perinatal outcomes; low offspring birth weight [START_REF] Andres | Perinatal complications associated with maternal tobacco use[END_REF][START_REF] Cnattingius | Decreasing smoking prevalence during pregnancy in Sweden: the effect on small-for-gestational-age births[END_REF][START_REF] Hardy | Does maternal smoking during pregnancy have a long-term effect on the child?[END_REF][START_REF]The 2004 United States Surgeon General's Report: The health consequences of smoking[END_REF][START_REF] Cnattingius | The epidemiology of smoking during pregnancy: smoking prevalence, maternal characteristics, and pregnancy outcomes[END_REF] , preterm birth [START_REF] Andres | Perinatal complications associated with maternal tobacco use[END_REF][START_REF]The 2004 United States Surgeon General's Report: The health consequences of smoking[END_REF][START_REF] Cnattingius | The epidemiology of smoking during pregnancy: smoking prevalence, maternal characteristics, and pregnancy outcomes[END_REF][START_REF] Kyrklund-Blomberg | Preterm birth and maternal smoking: risks related to gestational age and onset of delivery[END_REF] , spontaneous abortion, [START_REF]The 2004 United States Surgeon General's Report: The health consequences of smoking[END_REF][START_REF] Cnattingius | The epidemiology of smoking during pregnancy: smoking prevalence, maternal characteristics, and pregnancy outcomes[END_REF][START_REF] Ernst | Behavioral and neural consequences of prenatal exposure to nicotine[END_REF] and sudden infant death syndrome [START_REF]The 2004 United States Surgeon General's Report: The health consequences of smoking[END_REF][START_REF] Cnattingius | The epidemiology of smoking during pregnancy: smoking prevalence, maternal characteristics, and pregnancy outcomes[END_REF][START_REF] Ernst | Behavioral and neural consequences of prenatal exposure to nicotine[END_REF][START_REF] Haglund | Cigarette smoking as a risk factor for sudden infant death syndrome: a population-based study[END_REF] . In addition, long lasting behaviour problems have been suggested; for example, compared to unrelated controls, offspring who experienced SDP have increased risk of externalizing problems [START_REF] Onofrio | Smoking during pregnancy and offspring externalizing problems: an exploration of genetic and environmental confounds[END_REF] including attention-deficit/hyperactivity disorder, [START_REF] Ernst | Behavioral and neural consequences of prenatal exposure to nicotine[END_REF][START_REF] Onofrio | Smoking during pregnancy and offspring externalizing problems: an exploration of genetic and environmental confounds[END_REF] aggression, 10 criminality, [START_REF] Ernst | Behavioral and neural consequences of prenatal exposure to nicotine[END_REF][START_REF] Onofrio | Familial confounding of the association between maternal smoking during pregnancy and offspring criminality: A populationbased study in Sweden[END_REF] poorer general cognitive functioning [START_REF] Kafouri | Maternal cigarette smoking during pregnancy and cognitive performance in adolescence[END_REF] and academic and intellectual performance. [START_REF] Lundberg | Maternal smoking during pregnancy and intellectual performance in young adult Swedish male offspring[END_REF][START_REF] Lambe | Maternal smoking during pregnancy and school performance at age 15[END_REF][START_REF] Onofrio | A quasi-experimental approach of maternal smoking during pregnancy and offspring academic achievement[END_REF] Birth-related SDP outcomes (e.g., lower birth weight, preterm birth) appear causal, [START_REF] Cnattingius | The epidemiology of smoking during pregnancy: smoking prevalence, maternal characteristics, and pregnancy outcomes[END_REF] whereas emerging evidence suggests that the link to most studied long-term behaviours is confounded by other risks or unmeasured familial effects, such as shared environmental or genetic risks. [START_REF] Onofrio | Smoking during pregnancy and offspring externalizing problems: an exploration of genetic and environmental confounds[END_REF][START_REF] Onofrio | Familial confounding of the association between maternal smoking during pregnancy and offspring criminality: A populationbased study in Sweden[END_REF][START_REF] Kafouri | Maternal cigarette smoking during pregnancy and cognitive performance in adolescence[END_REF][START_REF] Lundberg | Maternal smoking during pregnancy and intellectual performance in young adult Swedish male offspring[END_REF][START_REF] Lambe | Maternal smoking during pregnancy and school performance at age 15[END_REF][START_REF] Onofrio | A quasi-experimental approach of maternal smoking during pregnancy and offspring academic achievement[END_REF][START_REF] Knopik | Maternal smoking during pregnancy and child outcomes: real or spurious effect?[END_REF] One aspect of individual development that recently received substantial interest is the ability to cope with stress. For instance, increased stress vulnerability has been observed as a consequence of prenatal nicotine exposure in rats. [START_REF] Vaglenova | An assessment of the long-term developmental and behavioral teratogenicity of prenatal nicotine exposure[END_REF] Animal studies also suggest that prenatal nicotine exposure increases locomotor activity and causes learning and memory problems. [START_REF] Ernst | Behavioral and neural consequences of prenatal exposure to nicotine[END_REF] Specifically, the foetal programming hypothesis [START_REF] Phillips | Programming of the stress response: a fundamental mechanism underlying the long-term effects of the fetal environment?[END_REF][START_REF] Phillips | Fetal programming of autonomic and HPA function: do people who were small babies have enhanced stress responses?[END_REF] includes suggestions that the major regulatory systems involved in stress responses, the autonomic nervous system and the hypothalamic-pituitary-adrenal axis, could be permanently altered early in life; both pre-and postnatally. [START_REF] Phillips | Programming of the stress response: a fundamental mechanism underlying the long-term effects of the fetal environment?[END_REF][START_REF] Phillips | Fetal programming of autonomic and HPA function: do people who were small babies have enhanced stress responses?[END_REF] These alterations could, for example, be caused by restricted access to food or specific nutrients, maternal adversity, or exposure to synthetic glucocorticoids (e.g. cortisol). [START_REF] Phillips | Fetal programming of autonomic and HPA function: do people who were small babies have enhanced stress responses?[END_REF] Birth weight has often been used as a proxy for measuring adverse foetal environment, [START_REF] Phillips | Programming of the stress response: a fundamental mechanism underlying the long-term effects of the fetal environment?[END_REF][START_REF] Phillips | Fetal programming of autonomic and HPA function: do people who were small babies have enhanced stress responses?[END_REF] Smoking during pregnancy and offspring stress coping 4 and prior Swedish studies suggested that offspring size at birth is related to the measure of stress susceptibility used in the current study. [START_REF] Nilsson | Increased susceptibility to stress at a psychological assessment of stress tolerance is associated with impaired fetal growth[END_REF][START_REF] Nilsson | Fetal growth predicts stress susceptibility independent of parental education in 161991 adolescent Swedish male conscripts[END_REF][START_REF] Lundgren | Intellectual and psychological performance in males born small for gestational age[END_REF] Other possible teratogenic SDP-related mechanisms include disturbed placenta function and impaired transport of nutrients and oxygen to the foetus, [START_REF] Ernst | Behavioral and neural consequences of prenatal exposure to nicotine[END_REF] and nicotine-caused intrauterine hypoxia or birth asphyxia [START_REF] Ernst | Behavioral and neural consequences of prenatal exposure to nicotine[END_REF] leading to foetal death or long-term neurological deficits, or cellular alterations to the central and peripheral nervous systems. [START_REF] Ernst | Behavioral and neural consequences of prenatal exposure to nicotine[END_REF][START_REF] Slotkin | Fetal nicotine or cocaine exposure: which one is worse?[END_REF] The decrease in "foetal breathing" (foetal thoracic movements), reported to occur after nicotine exposure, causes lung maturation to slow down and reduces the blood flow to the foetus. [START_REF] Ernst | Behavioral and neural consequences of prenatal exposure to nicotine[END_REF] Additionally, activation of neurotransmitter receptors in the foetal brain could lead to epigenetic alterations involving permanent change in cell functioning that might not be detected until much later in the more developed, adolescent, brain. [START_REF] Ernst | Behavioral and neural consequences of prenatal exposure to nicotine[END_REF][START_REF] Huizink | Maternal smoking, drinking or cannabis use during pregnancy and neurobehavioral and cognitive functioning in human offspring[END_REF] However, another important mechanism through which SDP could effect offspring behaviour is the passing of genetic vulnerability from parent to offspring; a passive gene-environment correlation. [START_REF] Rutter | Proceeding From Observed correlation to causal inference[END_REF] In effect, repeated results from studies of behavioural problems (e.g. externalizing behaviour, 9 criminality, [START_REF] Onofrio | Familial confounding of the association between maternal smoking during pregnancy and offspring criminality: A populationbased study in Sweden[END_REF] and poor academic achievement [START_REF] Lambe | Maternal smoking during pregnancy and school performance at age 15[END_REF][START_REF] Onofrio | A quasi-experimental approach of maternal smoking during pregnancy and offspring academic achievement[END_REF] ) in offspring exposed to SDP were later found to be entirely confounded by familial risks. That is, a selective mechanism for SDP exposure exists so that mothers who smoke during pregnancy share also other risk factors with their children; hence, these other risk factors cause the observed adverse outcomes rather than SDP per se. Variables such as maternal age, education, or socioeconomic status might be the source of this selection, [START_REF] Ernst | Behavioral and neural consequences of prenatal exposure to nicotine[END_REF] but also unmeasured familial environment similarity and genetic risks. We aimed to investigate whether the association between intrauterine exposure to SDP and stress coping in Swedish late adolescent men persisted after controlling for measured and unmeasured confounding caused by intrafamilial similarity.

Methods

Study population

We linked several nation-wide longitudinal registries, maintained by government agencies in Sweden, using the unique personal identification number given to all Swedish citizens. We used data from the Multi-Generation [START_REF]Multi-generation register 2005 -A description of contents and quality[END_REF] and Education Registers, [START_REF] Raudenbush | Hierarchical linear models -applications and data analysis methods[END_REF] the 1990 Swedish Census, [START_REF]Folk-och bostadsräkningen[END_REF] and the Conscript, [START_REF] Magnusson | Strong inverse association between height and suicide in a large cohort of Swedish men: evidence of early life origins of suicidal behavior?[END_REF] Medical Birth, [START_REF]The Swedish Medical Birth Register -A summary of content and quality[END_REF][START_REF] Cnattingius | A quality study of a medical birth registry[END_REF] Total Population, and National Crime Registers. [START_REF] Fazel | The population impact of severe mental illness on violent crime[END_REF] Eligible for the study were all male youth in Sweden who underwent an evaluation regarding suitability for duty by a clinical psychologist at compulsory conscription for military services during 1997-2006 and born 1982-1988 (SDP registration at antenatal care started in 1982).

For this study, we used data collected regarding stress-coping (n=187 106). Military conscription was mandatory for Swedish men until 2008 and enforced by law. The majority of conscripts were 18 years old (79.3%; mean age=18.2, standard deviation (SD)=0.4, median=18.2, range 17.1-24.3 years). Individuals were linked to their siblings and cousins via parents and grandparents using the Multi-Generation Register, thus identifying extended-and nuclear families. This register links all children born 1932 or later in Sweden to both their parents. Nuclear families were indexed by the mothers (164 563 mothers with at least one child), whereas extended families were indexed by the maternal or paternal grandmother. There were 150 268 extended families with at least one individual in the offspring generation.

Offspring with both maternal and paternal cousins could be included in two extended families. Starting in 1982, all pregnant women in contact with public tax-funded antenatal care in Sweden are asked by their personal midwife about SDP; this information is included in the Medical Birth Register. The coverage is excellent; over 98% of all births are recorded in the register. [START_REF]The Swedish Medical Birth Register -A summary of content and quality[END_REF] SDP data were available for 162 371 of the 187 106 (86.8%) pregnancies in the study. Of these, 44 550 women reported smoking during pregnancy (27.4%) which is comparable with earlier findings. [START_REF] Cnattingius | The epidemiology of smoking during pregnancy: smoking prevalence, maternal characteristics, and pregnancy outcomes[END_REF][START_REF] Onofrio | Smoking during pregnancy and offspring externalizing problems: an exploration of genetic and environmental confounds[END_REF][START_REF] Onofrio | Familial confounding of the association between maternal smoking during pregnancy and offspring criminality: A populationbased study in Sweden[END_REF][START_REF] Kafouri | Maternal cigarette smoking during pregnancy and cognitive performance in adolescence[END_REF] The validity of self-reported SDP is high in general [START_REF] Lindqvist | Smoking during pregnancy: comparison of self-reports and cotinine levels in 496 women[END_REF] and previous studies suggest good validity also in the current sample. [START_REF] Cnattingius | Decreasing smoking prevalence during pregnancy in Sweden: the effect on small-for-gestational-age births[END_REF][START_REF] Hatziandreu | The reliability of self-reported cigarette consumption in the United States[END_REF] Outcome A clinical psychologist rated individual psychological functioning (PF) at conscription, purportedly reflecting stress-coping during wartime [START_REF] Nilsson | Increased susceptibility to stress at a psychological assessment of stress tolerance is associated with impaired fetal growth[END_REF][START_REF] Leboeuf-Yde | Coping and back problems: analysis of multiple data sources on an entire cross-sectional cohort of Swedish military recruits[END_REF] based on a standardized, 20-25 minute semi-structured interview. PF was rated 1-9 on a nine-point Likert-type scale; higher values indicate better coping. The distribution was stipulated to be normal with mean=5 and SD=2 (χ 2 goodness-of-fit test with 9 categories; p-value=0.23, indicating no reason to reject the normality assumption). The individual PF score was used as a proxy for general ability to cope with stress.

Exposure

Covariates

We adjusted our analyses using offspring, nuclear family and extended family confounders and mediators.

Offspring confounders. Maternal age was divided into five categories; <20, 20-24, 25-29, 30-34 and >34 years. Birth year was used alone and together with Conscript Register data to stratify age at conscription into categories; ≤17.50, 17.51-18.50, and >18.50 years. A birth order variable for male nuclear family offspring was also constructed. Offspring mediators. We considered two mediators, both obtained from the Medical Birth Register: gestational time divided into categories; <32, 32-36, 37-41 and >41 weeks, and birth weight.

Parental confounders. Parental occupation, divided into seven categories [START_REF]Stockholm: Statistics Sweden[END_REF] , income, and cohabitation status were all based on the 1990 Census. The Register of Education for 2004 provided highest parental educational level,classified into 7 categories. [START_REF] Rutter | Proceeding From Observed correlation to causal inference[END_REF] Parental criminal convictions for 1973-2004 were collected from the National Crime Register. The Total Population Register supplied mother's country of birth divided into 12 categories according to geographic and demographic similarities. Finally, we included a variable indicating if a halfsibship existed for offspring within the nuclear family.

Extended family confounders. We also included a variable on whether an individual had maternal or paternal half-cousins.

Statistical methods

To analyze the effect of SDP on PF, we used linear regression treating PF as a normally distributed variable. Results are presented both crude (unadjusted) and adjusted for possible confounders. To handle possible period effects, we adjusted the crude unrelated and cousin models for birth year and the crude sibling model for birth order.

Since birth weight and gestational age could be mediators of the association between SDP and PF, additional analyses were run to investigated whether these covariates mediated the association between SDP and PF as proposed by the foetal programming hypothesis.

Since we aimed at isolating a possible direct effect of SDP on PF by eliminating possible familial confounding, we ran additional models to test if familial effects distorted the

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Smoking during pregnancy and offspring stress coping 8 association. With these, we compared PF in siblings and cousins differentially exposed to SDP to explore if the association remained when looking at within-family effects (i.e., if differentially exposed siblings/cousins also differed in PF). Hence, we used the extended family and nuclear family as clusters and sub-clusters to capture similarities within families.

These analyses were performed with hierarchical linear models (HLM) [START_REF] Raudenbush | Hierarchical linear models -applications and data analysis methods[END_REF][START_REF] Harden | A behavior genetic investigation of adolescent motherhood and offspring mental health problems[END_REF] using SAS Proc Mixed. [START_REF] Littell | SAS for mixed models[END_REF] Thus, unmeasured variables common to individuals in the nuclear or extended family (i.e., shared genes and environments) were accounted for. [START_REF] Onofrio | Smoking during pregnancy and offspring externalizing problems: an exploration of genetic and environmental confounds[END_REF] We call this approach the Children-of-Siblings model since it is similar to the statistical methods used when examining variables for children of twins in conjunction with variables for their parents, or the Childrenof-Twins model. [START_REF] Onofrio | Smoking during pregnancy and offspring externalizing problems: an exploration of genetic and environmental confounds[END_REF][START_REF] Harden | A behavior genetic investigation of adolescent motherhood and offspring mental health problems[END_REF][START_REF] Onofrio | A genetically informed study of marital instability and its association with offspring psychopathology[END_REF][START_REF] Onofrio | The role of the children of twins design in elucidating causal relations between parent characteristics and child outcomes[END_REF] SDP and continuous covariates were centred around the cluster means (for both nuclear-and extended family) which yielded covariates equivalent to fixed effects. [START_REF] Onofrio | A quasi-experimental approach of maternal smoking during pregnancy and offspring academic achievement[END_REF] Furthermore, this procedure reduced possible bias due to correlation between covariates and residual errors. [START_REF] Neuhaus | Separating between-and within-cluster covariate effects by using conditional and partitioning methods[END_REF] We utilized an informed backwards elimination process when deciding which covariates to use; thus, these may differ across models.

We performed sibling-sibling and cousin-cousin comparisons on two different data subsets.

Siblings were compared using a subset consisting of two siblings within a nuclear family.

Nuclear families were solely indexed by mothers, since 91% of children in Sweden stays with their mother when parents divorce or separate, [START_REF]Fakta om den svenska familjen[END_REF] Smoking during pregnancy and offspring stress coping 9 influence PF in full-and half-sibling/cousins we conducted the analyses separately. The concordance of SDP in sibling-and cousin pairs are presented in Table 1.

- ---------------------------------------------Insert Table 1 about here ----------------------------------------------Finally, we aimed to disentangle the source of the familial confounding of the association between maternal SDP and her boy's PF by partitioning the variance of the intergenerational association in a two-level hierarchical structural equation model (SEM) using the statistical software program Mplus. [START_REF] Muthén | Mplus technical appendices[END_REF][START_REF] Muthén | Mplus user's guide[END_REF] Analyses were first performed with PF, and then on the residuals of PF retrieved from a linear regression model using the covariates. Because the results of these two analyses were very similar, we present only the analyses of the former.

Results from the latter analyses are presented in the Supplemental Online Material (SOM; Figure S1). Wherever possible, we picked one pair of sisters and their children for each extended family (24 468 children from 11 485 sister-pairs). The two SEM levels refer to within-mothers, comparing the association between mothers' SDP and offspring's PF within each of the nuclear families, and between-mothers, comparing the average SDP association with the average SDP between nuclear families. The variance was partitioned into three parts A, C, and E corresponding to genetics, shared environment (makes siblings similar) and nonshared environment (makes siblings different) [START_REF] Onofrio | Smoking during pregnancy and offspring externalizing problems: an exploration of genetic and environmental confounds[END_REF] (Figure 1). The partitioning of the variance comes from taking into account the genetic relatedness between the mothers; full siblings share 50% of their co-segregating genes while half-siblings share 25%. This notion was incorporated in the SEM as constraints on the genetic variance parameter modelled (V A in Figure 1). Another constraint was that the modelled shared environment variance parameter

F o r R e v i e w O n l y
Smoking during pregnancy and offspring stress coping 10 V C was equal within full sibling pairs and maternal half-siblings, whereas this parameter was set to 0 for paternal half-siblings (again, because 91% of the children remain with their mother when parents separate). [START_REF]Fakta om den svenska familjen[END_REF] This way of modelling allows us to draw conclusions about which of the intergenerational paths (b A , b C and b E ) that explain the association between SDP and PF. The method, as applied in Children-of-Twins model, has been described elsewhere. [START_REF] Onofrio | Smoking during pregnancy and offspring externalizing problems: an exploration of genetic and environmental confounds[END_REF][START_REF] Onofrio | The role of the children of twins design in elucidating causal relations between parent characteristics and child outcomes[END_REF] - ---------------------------------------------Insert Figure 1 about here ----------------------------------------------

Results

Mean PF scores are presented in Table 2 for SDP and offspring covariates and in Table 3 for parental covariates.

- ---------------------------------------------Insert Tables 2 and3 about here ----------------------------------------------The crude estimate of the association of SDP with PF was -0.38 (95%CI, -0.40 --0.36, p-value<0.0001; Figure 2, for results from the regression analysis: SOM Table S1). This means that a child exposed to SDP on average had a PF or stress-coping score 0.38 points lower on the nine-point scale than an unrelated child unexposed to SDP. The following analyses explored the mechanisms behind this negative association. Each of the included covariates individually predicted PF (analyses not shown). When adjusting for these, the association was attenuated (b=-0.15; 95%CI, -0.18 --0.13, p-value<0.0001, Figure 2; SOM Table S2).

Having adjusted for known confounders, we investigated if the association was due to unmeasured familial confounding by performing sibling and cousin analyses with HLM. The crude and adjusted between extended family estimates ("unrelated" estimates) remained approximately similar (Figure 2). Thus, offspring in extended families where SDP had occurred also had lower mean PF scores, regardless of if a mother smoked during his own or somebody else's pregnancy (sibling's or a cousin's). As seen in Figure 2, family adjusted effects (from the sibling and cousin models) differed from "unrelated" effects. When accounting for covariates for SDP-discordant siblings, the within regression effect of SDP on PF completely disappeared (b=0.13; 95%CI, -0.01 -0.27, p-value=0.068, Figure 2), providing strong evidence for familial confounding. Another way to address the mechanism behind this would be to study also half-siblings, but because of too few half-siblings discordant for SDP (59 pairs, Table 1) the statistical power was minimal (SOM Table S6).

When full cousins were studied, the adjusted within regression parameter was close to zero (b=-0.05; 95%CI, -0.11 -0.01, p-value=0.073, Figure 2) and reduced compared to the within half-cousins effect (b=-0.23; 95%CI, -0.44 --0.02, p-value=0.030, Figure 2) and to the between extended family parameter, again indicating substantial familial confounding. For full tables of regression coefficients from sibling and cousin analyses see SOM Tables S3-S10.

We found minimal influence of birth weight and gestational age on the association between SDP and PF, suggesting that these covariates did not mediate neither the crude nor the Smoking during pregnancy and offspring stress coping 12 adjusted association that we could verify. This held true for unrelated comparisons as well as sibling and cousin comparisons (analyses not shown).

- ---------------------------------------------Insert Figure 2 about here --------------------------------------------- p-value=0.66), which indicates that one latent variance parameter in the model is negligible. [START_REF] Onofrio | The role of the children of twins design in elucidating causal relations between parent characteristics and child outcomes[END_REF] To test whether the ACE model fitted the data better than the AE-and/or the CE model, two scaled-difference χ 2 -tests [START_REF] Satorra | A scaled difference chi-square test statistic for moment structure analysis[END_REF] were performed. We found no evidence that the ACE model explained the data better than the AE model (χ 2 =1.2, degrees of freedom=2, p-value=0.56). In contrast, when comparing the ACE model with the CE model the result was in favour of the ACE model (χ 2 =11.2, df=2, p-value=0.004). We used the Bayesian Information Criterion (BIC) to determine which of the models ACE and AE that fitted the data best. The AE model outperformed the ACE (BIC AE =126 493, BIC ACE =126 511; The Akaike Information Criterion (AIC) yielded a similar result: AIC AE =126 277, AIC ACE =126 280). Therefore, in subsequent models the shared environment parameters were set to zero. [START_REF] Onofrio | Smoking during pregnancy and offspring externalizing problems: an exploration of genetic and environmental confounds[END_REF][START_REF] Onofrio | The role of the children of twins design in elucidating causal relations between parent characteristics and child outcomes[END_REF] Results from the model fitting are presented in Figure 3 ---------------------------------------------Insert Figure 3 about here ----------------------------------------------

Discussion

We aimed to investigate the effect of intrauterine exposure to SDP on offspring stress-coping in late adolescence. We used nation-wide longitudinal registers to compare not only unrelated individuals differentially exposed to SDP, but also relatives (siblings and cousins) to explore possible familial confounding and estimate the roles of genetic and environmental determinants.

Our main finding was that the observed association between SDP and poorer PF was entirely confounded by familial factors. Since the association could not be entirely explained by selected à priori confounders, we applied models that investigated unmeasured confounding based on similarities within nuclear and extended families. Familial confounding was evident; the association between SDP and poorer offspring stress-coping decreased when half-cousin comparisons were used instead of unrelated individuals, and disappeared completely in within full cousin-and full sibling comparisons. A possible reason for mothers to change smoking habits between pregnancies is if a life-altering event has occurred. However a Swedish study on whether smoking habit changes after an adverse pregnancy outcome found only modest effects on continued smoking in next pregnancy. [START_REF] Cnattingius | Will an adverse pregnancy outcome influence the risk of continued smoking in the next pregnancy?[END_REF] Thus, this is probably not a major reason for the familial confounding, especially since such effects are even less influential in the comparison between smoking discordant sisters. Additionally, data suggested that genetic effects entirely accounted for this familial confounding. The present results concur with previous studies in humans suggesting that associations between SDP and cognitive/behavioural outcomes in adolescent offspring are not causal but subject to Smoking during pregnancy and offspring stress coping 14 substantial familial confounding, [START_REF] Onofrio | Smoking during pregnancy and offspring externalizing problems: an exploration of genetic and environmental confounds[END_REF][START_REF] Onofrio | Familial confounding of the association between maternal smoking during pregnancy and offspring criminality: A populationbased study in Sweden[END_REF][START_REF] Kafouri | Maternal cigarette smoking during pregnancy and cognitive performance in adolescence[END_REF][START_REF] Lundberg | Maternal smoking during pregnancy and intellectual performance in young adult Swedish male offspring[END_REF][START_REF] Lambe | Maternal smoking during pregnancy and school performance at age 15[END_REF][START_REF] Onofrio | A quasi-experimental approach of maternal smoking during pregnancy and offspring academic achievement[END_REF] primarily due to genetic rather than environmental mechanisms. One possible mechanism is that mothers transmit smoking liability to offspring and offspring smoking influenced stress reactivity. We could not test this since no data on smoking were available for the conscripts. Regardless, to be informative, studies of the effect of SDP and other parental risk factors on offspring must take familial confounding into account.

We also tested the foetal programming hypothesis, the study of which often used low birth weight as a proxy for adverse foetal environment. [START_REF] Phillips | Programming of the stress response: a fundamental mechanism underlying the long-term effects of the fetal environment?[END_REF][START_REF] Phillips | Fetal programming of autonomic and HPA function: do people who were small babies have enhanced stress responses?[END_REF] When comparing siblings or cousins differently exposed to SDP, the inclusion of birth weight as a potential mediator of the link between SDP and offspring PF, the latter remained essentially unchanged. Thus, either the lowering of birth weight due to SDP is not in the same causal pathway as the effect on PF or the effect of low birth weight on PF is also due to familial confounding.

Our study had several strengths, particularly its size and longitudinal total population-based design with high coverage of exposure data and outcome. As supported by Swedish data, we assumed that children are primarily raised by their biological mothers when parents divorce or separate. [START_REF]Fakta om den svenska familjen[END_REF] Since PF was assessed by professionals employed by the Swedish armed forces, the rating was classified. Hence, although previous studies used PF as a stress-coping measure, [START_REF] Nilsson | Increased susceptibility to stress at a psychological assessment of stress tolerance is associated with impaired fetal growth[END_REF][START_REF] Nilsson | Fetal growth predicts stress susceptibility independent of parental education in 161991 adolescent Swedish male conscripts[END_REF][START_REF] Lundgren | Intellectual and psychological performance in males born small for gestational age[END_REF][START_REF] Leboeuf-Yde | Coping and back problems: analysis of multiple data sources on an entire cross-sectional cohort of Swedish military recruits[END_REF] we could not explicitly validate it. As indirect support, however, the test has been used for several decades, and it has been validated in that it correlates with military rank at the completion of military service. [START_REF] Carlstedt | Validering av inskrivningsprövningen mot vitsord från den militära grundutbildningen[END_REF] Regarding exposure, we used mothers' smoking status at prenatal care registration (approximately the first trimester) as SDP measure. Self-reported SDP might be less reliable in later years, since the stigma associated with smoking while pregnant has increased. However, studies support its validity, [START_REF] Cnattingius | Decreasing smoking prevalence during pregnancy in Sweden: the effect on small-for-gestational-age births[END_REF][START_REF] Lindqvist | Smoking during pregnancy: comparison of self-reports and cotinine levels in 496 women[END_REF][START_REF] Hatziandreu | The reliability of self-reported cigarette consumption in the United States[END_REF] (1982-1988), and we controlled for period effects when including birth year/birth order as a covariate. We cannot differentiate between prenatal only and prenatal plus postnatal smoking; therefore, the associations examined could be due to postnatal smoking as well as SDP. Another limitation of the study is that analyses was done in men, hence generalization to women cannot be assumed.

Finally, we want to stress that mothers' smoking during pregnancy is associated with numerous adverse outcomes, especially related to birth and infancy. [START_REF] Cnattingius | The epidemiology of smoking during pregnancy: smoking prevalence, maternal characteristics, and pregnancy outcomes[END_REF][START_REF] Johansson | Maternal smoking and infant mortality: does quitting smoking reduce the risk of infant death?[END_REF] However, our results add to the accumulating evidence that smoking during pregnancy have no, or only minor, long-term causal effects on offspring cognitive functioning. [START_REF] Onofrio | Smoking during pregnancy and offspring externalizing problems: an exploration of genetic and environmental confounds[END_REF][START_REF] Onofrio | Familial confounding of the association between maternal smoking during pregnancy and offspring criminality: A populationbased study in Sweden[END_REF][START_REF] Kafouri | Maternal cigarette smoking during pregnancy and cognitive performance in adolescence[END_REF][START_REF] Lundberg | Maternal smoking during pregnancy and intellectual performance in young adult Swedish male offspring[END_REF][START_REF] Lambe | Maternal smoking during pregnancy and school performance at age 15[END_REF][START_REF] Onofrio | A quasi-experimental approach of maternal smoking during pregnancy and offspring academic achievement[END_REF] Other factors accounts for the association, and in the case of stress-coping the confounds seem to be mainly of genetic origin. To conclude, mothers prone to SDP also transmit genes to their children, which cause poorer stress coping in the latter. Smoking during pregnancy and offspring stress coping 16

Key messages

• Foetal exposure to tobacco smoking is suggested to cause long term alterations to regulatory systems involved in stress response.

• We found an association between smoking during pregnancy and worse stress coping in male offspring at 18 years of age.

• This association is not causal but subject to substantial familial confounding • The familial confounding seems to be primarily of genetic origin. Smoking during pregnancy and offspring stress coping 27 ‡ Criminal convictions were obtained from the national crime registry; hence, there were no missing data. § The low prevalence of half-siblings was due to the short time interval (7 years, 1982-1988) during which both the index children and the half siblings had to be born, and that only male children with a score for psychological functioning at conscription were included.

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Smoking during pregnancy and offspring stress coping 29

  Smoking during pregnancy and offspring stress coping 5

  Smoking during pregnancy and offspring stress coping 6

  Smoking during pregnancy and offspring stress coping 7

  and our explicit aim was to capture possible familial effects. There were 26 118 individual siblings within 13 059 nuclear families. Cousin comparisons were made on a subset consisting of two cousins within an extended family.There were 52 888 individuals from 47 684 nuclear families within 26 421 extended families included in the subset (individuals eligible for comparisons within two extended families might be included twice in the analysis). To examine the difference between how SDP

  Smoking during pregnancy and offspring stress coping 11

-

  Because the HLM analyses indicated substantial familial confounding, we tried to estimate genetic and environmental effects on the association using SEM. When fitting the ACE model, both the intergenerational paths b C and b E had very large standard errors (b A =-1.48, standard error (SE)=0.90, p-value=0.10, b C =-1.31, SE=2.65 p-value=0.62; b E =0.18, SE=0.41

  . The regression coefficient b A expressing the genetic intergenerational transmission on PF was negative (b A =-1.44, SE=0.13, p-value<0.0001) while the non-shared environmental regression coefficient was positive (b E =0.44, SE=0.21, p-value=0.037). For the full model, see SOM Figure S2.

  Smoking during pregnancy and offspring stress coping 13 -

Smoking during pregnancy and offspring stress coping 28 Figure 1 .

 281 Figure 1. Variance partitioning for A, genetics; C, the shared environment; and E, non-shared

Figure 2 .

 2 Figure 2. Regression coefficient estimates and 95% confidence intervals for psychological functioning as a function of maternal smoking during

Smoking during pregnancy and offspring stress coping 30 Figure 3 .

 303 Figure 3. Variance partitioning for A, genetics; C, the shared environment; and E, non-shared

  the period when SDP was

	measured was quite short
	F o r
	R
	e v
	i e
	w
	O n l
	y

Table 1 .

 1 Concordance regarding exposure to maternal smoking during pregnancy in siblingand cousin pairs among all male children born 1982-1988 in Sweden and assessed for psychological functioning at age 18 as part of mandatory military conscript evaluation

	Relation	SDP = 0	SDP = 1	Total
	Full siblings			25 452
	Concordant	18 702	4450	23 152
	Discordant	1150	1150	2300
	Half siblings			666
	Concordant	256	292	548
	Discordant	59	59	118
	Full cousins			50 038
	Concordant	27 480	5106	32 586
	Discordant	8726	8726	17 452
	Half cousins			2850
	Concordant	978	492	1470
	Discordant	690	690	1380

Table 2 .

 2 Offspring characteristics for all 187 106 male children born 1982-1988 in Sweden and assessed for psychological functioning at age 18 as part of mandatory military conscript

	evaluation
	Characteristic	n (%)	Psychological
			functioning
			Mean (SD)
	Smoking during pregnancy
	Yes	44 550 (23.8)	4.6 (1.8)
	No	117 822 (63.0)	5.0 (1.8)
	Missing	24 734 (13.2)	4.9 (1.8)
	Gestational time, weeks
	28-31	654 (0.3)	4.5 (1.7)
	32-36	8966 (4.8)	4.8 (1.8)
	37-41	162 873 (87.4)	4.9 (1.8)
	>41	13 812 (7.4)	4.9 (1.8)
	Missing	801 (0.4)	4.7 (1.7)
	Age at conscription, years
	≤17.50	2245 (1.2)	5.0 (1.7)
	17.51-18.50	158 625 (84.8)	4.9 (1.8)
	>18.50	26 236 (14.0)	4.6 (1.9)
	Year of birth
	1982	29 971 (16.0)	4.8 (1.8)
	1983	28 231 (15.1)	4.9 (1.8)
	1984	25 915 (13.9)	5.1 (1.7)
	1985	30 243 (16.2)	4.8 (1.9)

Table 3 .

 3 Characteristics for parents of all 187 106 male children born 1982-1988 in Sweden and assessed for psychological functioning at age 18 as part of mandatory military conscript evaluation.

		Maternal characteristics	Paternal characteristics
	Characteristic	n (%)	Psychological	n (%)	Psychological
			functioning		functioning
			Mean (SD)		Mean (SD)
	Parent's occupation
	Unskilled blue collar	48 941 (26.2)	4.6 (1.7)	34 349 (18.4)	4.6 (1.8)
	worker
	Skilled blue collar	20 845 (11.1)	4.8 (1.7)	37 737 (20.2)	4.7 (1.7)
	Low level white collar	28 390 (15.2)	5.0 (1.7)	16 086 (8.6)	5.0 (1.8)
	Intermediate level white	36 426 (19.5)	5.2 (1.8)	32 521 (17.4)	5.1 (1.8)
	collar
	High level white collar	13 395 (7.2)	5.3 (1.8)	28 976 (15.5)	5.2 (1.8)
	Self employed	5615 (3.0)	5.0 (1.8)	14 889 (8.0)	4.9 (1.8)
	No	10 576 (5.7)	4.7 (1.8)	9692 (5.2)	4.7 (1.8)
	information/uncategorized
	Missing †	22 918 (12.2)	4.6 (1.8)	12 856 (6.9)	4.5 (1.8)
	Parent's income, Swedish
	kronor
	<100 000	91 964 (49.2)	4.7 (1.8)	25 538 (13.6)	4.5 (1.8)
	100 000-199 900	89 125 (47.6)	4.9 (1.8)	96 755 (51.7)	4.7 (1.8)
	200 000-299 900	5194 (2.8)	5.4 (1.8)	50 536 (27.0)	5.1 (1.8)
	300 000-399 900	686 (0.37)	5.4 (1.8)	10 059 (5.4)	5.4 (1.7)

Acknowledgements

This work was supported by the Swedish National Prison and Probation R&D; the Swedish Research Council -Medicine; the Indiana University Faculty Research Support Program, NARSAD; the National Institute of Child Health and Human Development [grant number HD056354]


Frank Van Oosterhout

Miquel Lurling
email: miquel.lurling@wur.nl

Effects of the novel 'Flock & Lock' lake restoration technique on Daphnia in Lake Rauwbraken (The Netherlands)

Keywords: cyanobacteria blooms, eutrophication, grazing inhibition, mitigation, zooplankton Cyanobacteria blooms, eutrophication, grazing inhibition, mitigation, 18 zooplankton

Effects of the novel 'Flock & Lock' lake restoration

technique on Daphnia in Lake Rauwbraken (The Netherlands)

'Flock & Lock' to Lake Rauwbraken, a small, isolated deep lake in The Netherlands, 74 that has suffered from frequent blooms of cyanobacteria during the last decade. 75

The mitigating action included the surface application of the modified clay. In 76 laboratory experiments, no detrimental effects of the active ingredient in the modified 77 clay, lanthanum, on the zooplankton grazer Daphnia magna could be detected 78 [START_REF] Lürling | Effects of Lanthanum and Lanthanum-modified 452 clay on growth, survival and reproduction of Daphnia magna[END_REF]. However, D. magna kept for 5 days at a realistic dosage 79 of the modified clay were 7-13% lighter than their conspecifics in controls (Lürling 80 and Tolman, 2010), which might suggest an effect of the clay carrier. Since filter 81 feeding zooplankton such as Daphnia are generalist feeders and thus do not 82 discriminate between food and clay particles, ingestion of clay can have detrimental 83 effects on the organisms. Several studies have revealed that suspended clays cause 84 decreased feeding, lower survival and reduced growth in Daphnia (McCabe and 85 O'Brien, 1983;[START_REF] Cuker | Type of suspended clay influences zooplankton 413 response to phosphorus loading[END_REF][START_REF] Kirk | Suspended day reduces Daphnia feeding rate: behavioural 435 mechanisms[END_REF][START_REF] Kirk | Effects of suspended clay on Daphnia body growth and fitness[END_REF][START_REF] Kirk | Suspended clay and the population dynamics of 439 planktonic rotifers and cladocerans[END_REF]86 Robinson et al., 2010). 87

To investigate the effects of the 'Flock & Lock' application on the Daphnia 88 population in Lake Rauwbraken, the lake was monitored before and after the 89 application, including a variety of water quality variables. In addition, we performed a 90 short-term grazing experiment with D. galeata (the main cladoceran in Lake 91 Rauwbraken), to test the hypothesis that the addition of the flocculent and/or the 92 modified clay reduces feeding activity. 93 94 METHODS 95

Lake Rauwbraken 96

The oval shaped Lake Rauwbraken is located in the south of The Netherlands (Fig. 1). 97 Lake Rauwbraken (Berkel-Enschot, The Netherlands) is a 4 ha, 15 m deep (maximum 98 Deleted: Lake Rauwbraken (Berkel-Enschot, The Netherlands) is a 4 ha, 15 m deep (maximum depth), thermally stratifying, former sand-excavation, dug in 1967. It is currently in use as a recreational area. From the mid 90-ies onwards an increase in cyanobacterial blooms was observed, culminating in a 4 month swimming ban in 2007. The dominating cyanobacteria were Planktothrix rubescens, Microcystis aeruginosa, Aphanizomen flos-aquae, Anabaena spp. and Woronichinia naegeliana. The first species was present in the lake whole year round, during summer months mostly in the deeper water layers, while the others were prevalent mostly during the summer months in the upper water layers.

Deleted:

In laboratory experiments, no detrimental effects of the active ingredient in the modified clay, lanthanum, on the zooplankton grazer Daphnia magna could be detected [START_REF] Lürling | Effects of Lanthanum and Lanthanum-modified 452 clay on growth, survival and reproduction of Daphnia magna[END_REF]. However, D. magna kept for 5 days at a realistic dosage were 7-13% lighter than their conspecifics in controls [START_REF] Lürling | Effects of Lanthanum and Lanthanum-modified 452 clay on growth, survival and reproduction of Daphnia magna[END_REF].

Deleted: Also suspended clay particles are known to cause feeding inhibition, lower survival and growth in Daphnia [START_REF] Kirk | Suspended clay and the population dynamics of 439 planktonic rotifers and cladocerans[END_REF][START_REF] Kirk | Suspended day reduces Daphnia feeding rate: behavioural 435 mechanisms[END_REF]. On the first day, the lake was de-phosphatised (removing phosphate) by applying 2 110 tons of a lanthanum modified clay (Phoslock®,provided by Bentophos GmbH,111 Ottersberg, Germany). The second day 2 tons of flocculent (Polyaluminiumchloride,112 PAC39 provided by Bentophos GmbH), pH buffered by 75 kg Ca(OH) 2 ,was applied 113 to remove all phytoplankton from the water column (flocculation, 'Flock'). On the 114 third day, 16 tons of modified clay were applied as sediment capping, to counteract P-115 efflux ('Lock'). All compounds were applied at the water surface from a barge, 116 through a spray manifold after mixing with lake water, thus the modified clay was 117 applied as a slurry, and the flocculent was strongly diluted. 118 119

Deleted: reveal

Field sampling 120

In the south-western centre of the lake, 65 m from the shore (51 o 34′55.35″ N, 121 5 o 07′52.14″ E), water samples of 1 or 2 L were taken by pump and hose (Reich,12 V 122 Tauch pumpe) over the entire water column at one meter intervals, which at this 123 1). Prior to the application, 124 samples were taken monthly from January to April 2008, then daily in the period from 125 three days prior to seven days after the application, followed by a biweekly sampling 126 interval in 2008 and 2009. The zooplankton was collected by filtering the water 127 through a 100 µm net, followed by fixation with Lugol's fixative, kept refrigerated 128 and dark. Daphnia were counted under a dissecting microscope at 15× magnification. 129 Also included are samples taken biweekly from April -June 2006 and2007. 130 Additional water samples were taken for measurement of water quality 131 variables such as chlorophyll-a concentration, suspended solid concentrations, 132 turbidity, metal concentrations and pH. On site pH was determined at one meter 133 intervals from the surface to 10 m depth using a WTW multi 350 I meter. Turbidity 134 was measured using a Hach 2100P turbidimeter. Chlorophyll-a was determined 135

following the ethanol extraction spectrophotometric method with phaeopigment 136 correction as described by [START_REF] Moed | Some problems in the estimation of 461 chlorophyll-a and phaeopigments from pre-and post-acidification 462 spectrophotometric measurements[END_REF]. Suspended solids were 137 measured after drying filters for 24 h at 105°C following the Dutch standard 138 procedure NEN-6484. Filtered (0.45 µm) and non-filtered (total) aluminium and 139 lanthanum concentrations were determined by ICP-MS in the Chemical-Biological 140 Soil Laboratory of the Department of Soil Sciences (Wageningen University). 141

The water quality variables chlorophyll-a, turbidity, pH and filterable-and 142 total aluminium and lanthanum concentrations through the water column were pooled 143 and grouped in periods before (day -10 to day 0), during (day 0 -3 ) and after the 144 application (day 4 -18). These pooled data were analyzed by one way ANOVA 145 followed by a Tukey post-hoc test or a non-parametric Kruskal-Wallis test using the 146 statistical tool-pack PAWS Statistics 17.0.3 (SPSS Inc). 147

Grazing experiment 149

The effects of the flocculent ('Flock') and the modified clay ('Lock') on the grazing 150 activity of D. galeata were examined in a short-term grazing experiment. Similarly 151 sized D. galeata (1.9 mm ± 0.1 mm; n =40) were placed in filtered (0.45 µm) water 152 from Lake Rauwbraken to which the green alga Scenedesmus obliquus was added as 153 food. 'Flock' and 'Lock' were tested separately and combined using the dosages as 154 applied in Lake Rauwbraken, i.e. 9 mg 'Flock' L -1 and 67 mg 'Lock' L -1 ; an 155 additional series was included with 10 times the 'Lock' concentration, since the 156 addition as a slurry to the surface of the water column could also have implied a 157 temporarily higher clay concentration. 158

The animals were collected from a stock culture, maintained in the laboratory 159 and fed three times a week with S. obliquus. The green alga S. obliquus SAG 276/3a 160 originated from the culture collection of the University of Göttingen (Germany). S. 161 obliquus was maintained in a 1.0 L chemostat system in continuous light of 120 µmol 162 quanta m -2 s -1 at 20°C on a slightly modified WC medium (Lürling and Beekman, 163 1999) and with a dilution rate of 1.1 d -1 . Prior to the experiment animals were selected, 164 rinsed with filtered Lake Rauwbraken water and placed in Lake Rauwbraken water 165 for three hours. Then individual animals were allocated to one of five treatments in a 166 well in 24 well culture plates. Each well held 2.5 mL food suspension in lake water 167 containing, no additive (controls) only flocculent ('Flock'), only P-fixative ('Lock') 168 both flocculent and P-fixative ('Flock & Lock') and ten times the dose of the mean in 169 situ treatment (10× 'Lock'). Each treatment was run in eight replicates, while four 170 wells per food treatment without a D. galeata served as algal controls. The well plates 171 were incubated for 3-4 h at 22°C in the dark. The clearance rates were calculated from 172

( ) ( ) [ ] 1 5 . 2 ln ln × × ∆ - = t CHL CHL CR daphnia ctrl
175 in which CR = the clearance rate (mL ind -1 h -1 ), CHL ctrl = the chlorophyll-a 176 concentration (µg L -1 ) in the control wells after an incubation period t, CHL daphnia = 177 the chlorophyll-a concentration (µg L -1 ) in the treatments wells after an incubation 178 period t (h), 2.5×1 = the amount of medium per individual D. galeata (mL ind. -1 ). 179

Clearance rates were compared using one way ANOVA followed by a Tukey's post-180 hoc comparison test. At the end of the experiment, body-length of each experimental 181 animal was measured from just above the eye to the base of the tail spine using a 182 dissecting microscope at 40× magnification. 183 184

Survival experiment 185

Since the application was expected to cause a strong reduction in phytoplankton 186 concentration up to 90% [START_REF] Schumaker | Zooplankton responses to 491[END_REF], a survival experiment with D. 187 galeata was performed. Animals were placed in 1 L jars that contained 800 mL 0.45 188 µm filtered Lake Rauwbraken water (No food), lake water with 2 µg L -1 CHL S. The 'Flock & Lock' application resulted in a strong reduction in chlorophyll-a 201 concentrations (Fig. 2). When chlorophyll-a concentrations over the water column 202 were pooled and grouped in periods before (day -10 to day 0), during (day 0 -3 ) and 203 after the application (day 4 -18), one way ANOVA (over natural-log transformed 204 data to fulfil homogeneity requirements) indicated significant differences (F 2, 173 = 205 121.7; p < 0.001). A Tukey post-hoc comparison revealed that chlorophyll-a 206 concentrations in each period were significantly different from each other, with the 207 highest concentrations of 12 ± 10 µg L -1 (average ± 1SD) before the application and 208 the lowest concentrations of 2 ± 1 µg L -1 after the application (Fig. 3). Water column 209 CHL concentrations before and during the application excluded the surface scum of 210

Aphanizomenon flos-aquae that was present in the south-western part of the lake with 211 CHL concentrations of 8 mg L -1 ; and which was precipitated effectively by the 212 treatment. Despite a significant reduction in CHL concentrations, addition of the 213 modified clay caused increased turbidity during the application (Fig. 4). The turbidity 214 before and after the application was similar (Table 1), but during the application 215 turbidity increased significantly (Kruskal-Wallis test: χ 2 = 16.8; p < 0.001).

216

The pH-values of the water before, during and after the application were 217 significantly different (Kruskal-Wallis test: χ 2 = 17.6; p < 0.001). Although the pH-218 values in all three periods were significantly different from each other, all values were 219 close to neutral (Table 1). The mean water column total phosphorus concentration was 220 10 reduced significantly (χ 2 = 24.9; p < 0.001) from ≈ 91 µg P L -1 before to ≈ 19 µg P L -1 221 after the application (Table 1). The suspended solid concentrations before the 222 application were significantly lower (χ 2 = 8.83; p = 0.012) than those found during 223 and after the application (Table 1). Filterable and total aluminium concentrations were 224 lowest before the application, highest during the application and then declined again 225 (Table 1). The maximum aluminium concentrations measured were 63.4 and 3030 µg 226 L -1 for filterable and total aluminium, respectively. Filterable and total lanthanum 227 concentrations were very low before, but considerably higher during and after the 228 application (Table 1). 229 230Daphnia in Lake Rauwbraken 231

The zooplankton grazer D. galaeta is a common inhabitant of Lake Rauwbraken and 232 was also present before and during the 'Flock & Lock' treatment in 2008 (Fig. 5). 233 However, about one week after the application, Daphnia had disappeared from the 234 water column, which had not been observed in the same periods in 2006, 2007and 235 2009 (Fig. 5 (Fig. 5). This disappearance points strongly towards a treatment effect. 236 Daphnids reappeared after several months where some D. galeata were observed in 237 the upper water layers, but the most remarkable observation was the explosion of D. 238 pulex in late summer, early autumn in the deep water layers (Fig. 6). In 2009, mostly 239 D. galeata were observed (Fig. 6). 240 241

Grazing experiment 242

The effect of the 'Flock & Lock' treatment on the feeding activity of D. galeata was 243 tested in a short-term grazing experiment. The water quality variables (mean ± 1SD) 244 were similar among treatments: pH 7.5 (± 0.1), conductivity 179 (± 5) µS cm -1 and 245

Deleted: strongly among the treatments (F 4,35 = 0.92; p = 0.461) and on average (± 1SD) 1.9 (± 0.1) mm. 247

The grazing activity as measured by the clearance rate (CR) was significantly affected 248 (F 4,35 = 69.1; p < 0.001) by the different treatments (Fig. 7). A Tukey test revealed 249 that CR on S. obliquus in controls was similar to those in the 'Lock' treatment, but CR 250 were significantly depressed in the 'Flock', the 'Flock & Lock' and the '10× Lock' 251 treatments (Fig. 7) as compared to both Control and Lock treatments . 252

253

Survival experiment 254

Animals in the highest food treatment (20 µg L -1 CHL) also had the highest survival 255 (Fig. 8). About 75% survived until the end of the experiment, while this was about 256 40% in the low food (2 µg L -1 CHL) and no food treatments (Fig. 8). Death rates were 257 significantly different (F 2,6 = 11.4; p = 0.009) and two homogenous groups could be 258 distinguished: 1) No food and the 2 µg L -1 CHL treatments with mean (± 1SD) death 259 rates of 0.40 (± 0.06) d -1 and 0.32 (± 0.12) d -1 , respectively, and 2) the 2 µg L -1 CHL 260 and 20 µg L -1 CHL treatments. Death rates in the later were 0.13 (± 0.02) d -1 . 261

262

DISCUSSION 263

The 'Flock & Lock' method as applied to Lake Rauwbraken, caused a temporary 264 disappearance of Daphnia in this lake. No Daphnia were found in water samples for 265 at least three months after the application, after which daphnids recovered. Several 266 factors may have caused the disappearance of D. galaeta following this treatment. 267 First, animals might have been trapped in the aluminium hydroxide flocks and 268 precipitated to the sediment [START_REF] Minzoni | Effects of aluminium on different forms of phosphorus and 459 freshwater plankton[END_REF][START_REF] Schumaker | Zooplankton responses to 491[END_REF]. This possibility 269 is, however, not supported by the field observations as animals, and especially 270 The addition of a low amount of the buffering agent Ca(OH) 2 (making 297 approximately 0.25 mg L -1 ) probably had no effect on Daphnia, as lime applications 298 with Ca(OH) 2 up to 78 mg L -1 had no effect on Daphnia [START_REF] Prepas | Long-term effects of successive Ca(OH) 2 and CaCO 3 treatments on 473 the water quality of two eutrophic hardwater lakes[END_REF]Zhang et 299 al., 2001). 300

In a recent study, Daphnia was exposed to the modified clay and its active 301 ingredient lanthanum [START_REF] Lürling | Effects of Lanthanum and Lanthanum-modified 452 clay on growth, survival and reproduction of Daphnia magna[END_REF]. That study yielded a no-observed 302 effect concentration (NOEC), based on reproduction, of 100 µg La L -1 , which was 303 similar to the result found in a 21 d Daphnia reproduction test [START_REF] Sneller | Maximum 496 permissible concentrations and negligible concentrations for rare earth 497 elements (REEs)[END_REF]. 304

The maximum concentration of filterable lanthanum in Lake Rauwbraken (90.8 µg L -1 ) 305 was close to the NOEC. Taking among species variability of sensitivity to lanthanum 306 into consideration, effects on reproduction of D. galeata can not be excluded. The 307 increases in juveniles during and immediately after the application were most likely 308 from broods that had been deposited in the animal's brood pouch prior to the 309 treatment of the Lake. 310

When exposed to the modified clay, somatic growth of juvenile Daphnia was 311 affected at concentrations exceeding 100 mg clay L -1 , while at 100 mg clay L -1 312 animals were already slightly lighter (7 -13%) than their conspecifics in controls and 313 treatments up to 50 mg L -1 [START_REF] Lürling | Effects of Lanthanum and Lanthanum-modified 452 clay on growth, survival and reproduction of Daphnia magna[END_REF]. These values are close to the 314 whole-lake averaged dosage of 67 mg L -1 . However, the application takes place 315 through a spray manifold where the slurry is brought into the upper water layer (Robb 316 et al., 2003). Hence, modified clay concentrations during and shortly after the 317 addition in the upper water layers will be much higher than the ones calculated over 318 the whole water body. This was also reflected in turbidity-depth profiles during and 319 [START_REF] Haghseresht | A Revolution in Phosphorous Removal[END_REF]Ross and 321 Cloete, 2006). Nevertheless, the short period (1 -2 days) of high turbidity (with 322 maxima of around 18 NTU) might have affected the feeding rate of the animals, as 323 shown by our grazing experiment, in which a clay dosage of ten times the whole-lake 324 averaged one, caused a strong feeding inhibition. This is corroborated by studies that 325 found strongly depressed feeding rates in Daphnia by suspended clay [START_REF] Kirk | Suspended day reduces Daphnia feeding rate: behavioural 435 mechanisms[END_REF]) 326 and at turbidities above 10 NTU [START_REF] Mccabe | The effects of suspended silt on feeding 455 and reproduction of Daphnia pulex[END_REF]. However, whether 327 short exposures to relatively high clay concentrations, and consequently low food 328 intake, have strong impact on the Daphnia population is not clear (Robinson et al., 329 2010). 330

Then it should also be noted that the turbidity before the application was 331 mainly a result of phytoplankton, whereas after the application it was mostly fine clay. 332

Reductions in juvenile Daphnia survival and growth by these clay concentrations are 333 plausible [START_REF] Kirk | Suspended clay and the population dynamics of 439 planktonic rotifers and cladocerans[END_REF], but also the generally low food concentrations 334 themselves seem to be a major factor in the disappearance of D. galeata. This is 335 corroborated by the significantly reduced survival of D. galeata in the low food 336 treatments (2 µg L -1 CHL) that resemble the post treatment concentrations in situ 337 compared to the pre treatment concentrations (∼20 µg L -1 CHL). 338

Interestingly, similar observations on Daphnia abundances have been made in 339 Newman Lake (Washington, USA) following an aluminium treatment, where two 340 weeks after the treatment especially cladocera, such as Daphnia disappeared, but 341 recovered in the months afterwards [START_REF] Schumaker | Zooplankton responses to 491[END_REF]. In Lake Rauwbraken, 342 recovery took longer which might be explained by the high predation pressure of 343 visually hunting fish in the crystal clear water during the spring and summer months. 344

Deleted: probably Deleted: had reduced Deleted: [START_REF] Kirk | Suspended day reduces Daphnia feeding rate: behavioural 435 mechanisms[END_REF], which is corroborated by the grazing experiment where a clay dosage of ten times the whole-lake averaged one caused a strong feeding inhibition. In October large-bodied D. pulex appeared in the lake, and also D. 347 galeata reappeared, probably since biomass of the main visually hunters 0 + -fish had 348 decreased substantially by then [START_REF] Van Gool | relationship between fish kairomone 504 concentration in a lake and phototactic swimming by Daphnia[END_REF]. 349

The 'Flock & Lock' treatment has been developed for restoring degraded or 350 eutrophied systems by precipitating particulate algal matter and phosphorus followed 351 by immobilizing phosphate permanently, and thereby reducing blooms of nuisance 352 cyanobacteria. In Lake Rauwbraken a bloom of Aphanizomenon flos-aquae and deep 353 water abundant Planktothrix rubenscens could be removed effectively. It is generally 354 accepted that such cyanobacteria cause major disruptions of the aquatic ecosystem 355 and that cyanobacteria have strong negative effects on Daphnia [START_REF] Lampert | Rare earth and yttrium phosphate solubilities in 444 aqueous solution[END_REF]356 Lürling, 2003). It has been suggested that a 'Flock & Lock' treatment probably would 357 lead to only minor effects on the Daphnia population, which would be outweighed by 358 significant reductions in the amount of cyanobacteria [START_REF] Lürling | Effects of Lanthanum and Lanthanum-modified 452 clay on growth, survival and reproduction of Daphnia magna[END_REF]. 359 However, the treatment resulted in a very strong effect on Daphnia, exemplified by 360 the spring data for the different years (see Fig. 5). Despite the presence of a persistent 361 bloom of P. rubescens in 2007, Daphnia remained present albeit in low numbers, 362 while in 2008 daphnids were eradicated. Daphnia are key organisms in the trophic 363 energy flow and comprise a major link between limnetic primary production and 364 higher trophic levels [START_REF] Mcqueen | Trophic relationships in 457 freshwater pelagic ecosystems[END_REF]. Although the efficiency of energy 365 transfer can be noticeably decreased when pelagic cyanobacteria start to flourish 366 [START_REF] Paerl | A comparison of cyanobacterial bloom dynamics in freshwater, 464 estuarine and marine environments[END_REF], the 'Flock & Lock' treatment probably had an even stronger effect. It 367 might explain why scuba-diving surveys did not report any 0 + -perch (Perca fluviatilis), 368 which were the most abundant fish in the previous years, whereas in the abundant 369 were abundant. In 2009, D. galeata was present in spring and also juvenile perch were 371 observed, which was supported by fyke catches of 1 + -perch in 2010. Gut contents of 372 several of these animals revealed they had been foraging on Daphnia. Hence, 373

Daphnia recovered from the treatment as has been observed in other lakes following 374 lake restoration [START_REF] Schumaker | Zooplankton responses to 491[END_REF] and represented an important food item for 375 juvenile fish. 376

The "Flock & Lock" treatment differs from classical lake restoration 377 techniques using only iron or aluminium salts, in the sense that here 378 polalumimiumchloride was used to flock particles, while the modified clay was 379 applied to give the flocks ballast and more importantly to intercept any phosphates 380 released from the precipitated flocks and underlying sediment. The active ingredient 381 in the clay, lanthanum, has a strong affinity for orthophosphate, forming the mineral 382 rhabdophane with an extreme low solubility product K sp = 10 -24.7 to 10 -25.7 mol 2 l -2 383 (Johanneson and Lyons, 1994;Liu and Byrne, 1997). Since this binding is not 384 influenced by redox state and a wide range of pH values, the rhabdophane formed can 385 be considered insoluble. However, pH can influence the absorption capacity of the 386 modified clay, as at pH > 9 hydroxyl ions compete with phosphate for lanthanum 387 binding-sites (Ross et al., 2008). The optimal pH-range for phosphate removal by 388 lanthanum is between 6 and 9 [START_REF] Peterson | Nutrient 469 inactivation as a lake restoration procedure -laboratory investigations[END_REF]Ross et al., 2008), where 389 phosphates bound to lanthanum can be considered to be immobilized permanently. It 390 is expected that Lake Rauwbraken will remain in the new oligo-mesotrophic state for 391 at least 10 years, since the dosage applied took the potentially bioavailable P in the 392 top 10 cm of the sediment and groundwater inflow into consideration. 393

  thermally stratifying, former sand-excavation, dug in 1967. It is currently used 99 as a recreational area. From the mid 1990s onwards an increase in cyanobacterial 100 blooms has been observed, culminating in a 4 month swimming ban in 2007. The 101 dominating cyanobacteria were Planktothrix rubescens, Microcystis aeruginosa, 102 Aphanizomenon flos-aquae, Anabaena spp. and Woronichinia naegeliana. The first 103 species was present in the lake year round, during summer months mostly in the 104 deeper water layers, while the others were prevalent mostly during the summer 105 months in the upper water layers. 106 107 Flock & Lock application 108 The Flock & Lock treatment was applied on three days, from April 21 st to 23 rd 2008. 109

  the surface to 10 m depth (Fig.

  chlorophyll-a derived from dark-adapted fluorescence using a

  189 obliquus and with 20 µg L -1 CHL. Food concentrations were checked after 4 and 8 190 days using a PHYTO-PAM and readjusted to the desired concentrations. Each 191 treatment was replicated three times and each jar received ten non-egg bearing D. 192 galeata from the stock cultures. After 1, 4, 8, 9 to 15 days jars were inspected for 193 survival. In the case of reproduction neonates were removed. Death rates were 194 calculated from linear regressions on natural-log transformed survival data. The death 195 by one-way ANOVA and significantly different means were 196 distinguished by a Tukey post-hoc comparison.

  5) o C. The body size of the experimental animals was similar 246

  during and the few days after the application. The disappearance 271 of D. galeata about one week after application also rules out acute toxicity of the or indirectly through efficient enveloping of algae which were then no longer 295 available to the Daphnia. 296

  application (data not shown). Turbidity declined rapidly as a 320 consequence of sedimentation of the clay particles

  (mainly Elodea nuttallii) 0 + -tench (Tinca tinca) 370

  the effects of the innovative 'Flock & Lock' lake restoration 394 technique on Daphnia in Lake Rauwbraken seemed to be caused by several factors 395 acting in synergy. Physical effects of flocks, grazing inhibition of flocks and clay, 396 very low food concentrations, absence of a predation refuge might all have played a 397 role. However, effects were temporary and Daphnia recovered from the treatment. , with an emphasis on cyanobacteria. Sci. World 1, 76-of Newman Lake, Washington. J.Freshw. Ecol.,

Fig. 1 .Fig. 4 .Fig. 6 .

 146 Fig. 1. Map showing Lake Rauwbraken (4 ha, mean depth 6 m, max depth 15 m) in 532 The Netherlands ( ) including the sampling station ( , 51 o 34′55.35″ N, 5 o 07′52.14″ 533

TABLE LEGENDS 515 516 Table 1 .

 LEGENDS5161 Water column averages (± 1 SD) of turbidity (NTU), pH, total phosphorus 517

	518	concentration (Total P, µg L -1 ) suspended solids (SS, mg L -1 ), filtered and non-filtered
	519	(total) aluminium and lanthanum (µg L -1 ) before, during and after the 'Flock & Lock'
	520	application in Lake Rauwbraken.
		Variable	Before application During application After application
		NTU	2.7 (3.1)	4.4 (4.2)	2.6 (1.3)
		pH	7.7 (0. 8)	7.3 (0. 6)	7.0 (0.3)
		Total P (µg L -1 )	91 (90)	34 (12)	19 (17)

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'Flock & Lock' treatment. Acute toxicity of the active ingredient in the flocculent,

143-192.

ACKNOWLEDGEMENTS 400

This study was supported by the Water board De Dommel and the Province Noord-401 Brabant. 402 403