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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26/01/2022 - 02/03/2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
(2013)
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
N/A
Analytical monitoring:
yes
Details on sampling:
Sampling schedule

Samples of test media including control group and solvent control group were taken from alternating test replicates and the mixing chamber supply of these replicates on study days -1, 0, and weekly thereafter until end of exposure. The changing intervals of the stock solution were considered.

Sampling and pre-treatment

At each sampling day 2 samples were taken per (alternating) test replicate. For each sample at least 30 mL of each test item concentration, the solvent control and the control were sampled.
For analysis of fresh and aged stock solutions, approx. 3 mL of each stock solution were sampled.

Vehicle:
yes
Remarks:
With regard to the limited solubility of the test item in water, ethanol (VWR, 99.97%, batch 21DO014013) was used as a solvent. The solvent concentration was the same in all concentration levels and the solvent control (0.025 mL/L).
Details on test solutions:
Stock solution

A stock solution of 1120 mg/L was prepared in ethanol. An appropriate amount of the test item was pipetted into a glass flask and filled up with the solvent. Density of the test item was taken into account. The solution was agitated until it was visually clear dissolved.
The stock solutions were prepared in appropriate intervals of 7 days.
Syringes were filled with the freshly prepared stock solutions or pure ethanol for the solvent control in corresponding intervals.
Details are given in Table 3 (attached).

Control

Dilution water (without test item and without solvent).

Solvent control

A solvent control with the same concentration of solvent but without test item was prepared and tested under the same conditions as the test groups.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Origin

Fish were bred at the test facility from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin, Germany)

Maintenance of brood fish

A breeding stock of unexposed, mature zebrafish aged between 9 and 11 months was used for egg production. Fish were free of macroscopically discernable symptoms of infection and disease. Spawners were maintained in aquaria with a loading capacity of a minimum of 1 L water per fish.

Spawning
15 – 35 adult zebrafish were kept in at least 3 separate aquaria. The fish were healthy with a mortality rate < 5% during the last 7 days and thus not medically treated for at least 7 days. About 15 minutes before artificial dawn, rectangular dishes covered with a stainless-steel mesh and containing artificial plants (plastic), were introduced into the aquaria. After 1 hour the glass dishes were removed. Any unhealthy eggs, as well as coagulated and un-fertilized eggs, were discarded (less than 30%). About 900 eggs were taken and washed in dilution water. Eggs originated from 3 different spawnings.
Test type:
flow-through
Water media type:
freshwater
Remarks:
Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Limit test:
yes
Total exposure duration:
35 d
Remarks on exposure duration:
(30 days post-hatch)
Post exposure observation period:
N/A
Hardness:
10 – 250 mg CaCO3/L
Test temperature:
26 ± 1.5 °C
pH:
6.0 – 8.5
Dissolved oxygen:
The dissolved oxygen concentrations in the control, solvent control and the test item groups, expressed in percent saturation, were in the mean 94 - 96% and ranged from 84 to 100% during the exposure period (see Table 13 and Table 14).
Salinity:
N/A
Conductivity:
172 µS/cm (recent measurement: 2021-11-09)
Nominal and measured concentrations:
The test was performed with three nominal test concentrations of 7.00 - 14.0 - 28.0 µg test item/L, which corresponded to arithmetric mean measured concentrations 4.72 - 8.45 - 13.9 µg/L of the dispersed fraction of the test item.
Details on test conditions:
Test design

A randomized block design with each treatment being present in each block was established. A flow-through exposure design was carried out. Membrane piston pumps provided the water flow-through. Precision syringe pumps were used for the introduction of stock solution. The stock solution and the dilution water were mixed in a mixing chamber (approx. volume 0.7 L) by magnetic stirring before passing the test aquaria (approx. volume 7.5 L; four replicates per test concentration, control and solvent control) where the eggs/fish were exposed. Glass tubes were used.
The accuracy of the water flow-through was checked prior to start of the exposure and three times per week thereafter. Water exchange in the test aquaria was about 10 times per day (3.125 L/h).
An equilibration period of at least 3 days was carried out prior to start of the exposure until measured concentrations of the test item showed no trend of increasing or decreasing levels.

Equilibration period

Test solutions flowed through the test vessels for 22 days prior to the start of the exposure until measured concentrations of the test item no trend of increasing or decreasing.

Control

Dilution water (without test item and without solvent)

Solvent control

Additionally, a solvent control with the same concentration of solvent but without test item was prepared and tested under the same conditions as the test groups.

Test duration

35 days (30 days post hatch), depending on post-hatch day 0 (study day 5).

Replicates, number of eggs

Four replicates per test concentration and control, with 20 eggs each (80 eggs per test concentration, solvent control and control) were tested.
For the whole study (including the range finding test and definitive test) 467 fish were used.

Loading

A loading rate not exceeding 0.5 g/L wet weight fish per 24 hours and not exceeding 5 g/L of solution at any time was maintained.

Test vessels

Glass aquaria of 8.7 L provided with mesh coated fittings allowing flow-through of test media (dimensions: 22/22/18 cm) were used. Test vessels were covered by glass lids. The volume of the test media was approximately 7.5 L.

Cleaning

The test vessels were siphoned as needed to remove excess fecal material and uneaten food, and to minimize microbial growth and biodegradation of the test item.
The mesh coated fittings were cleaned daily after start of feeding. Cleaning started on study day 5.

Aeration

The dilution water supply tank was aerated.
No additional aeration was provided.

Feeding of test fish

The feeding regime was ad libitum during the whole feeding period (study day 5 to 34).
Feeding started 3 days after the beginning of hatch on study day 5 (post-hatch day 1, where almost all non-affected larvae swum up). Larvae were fed with starter food (ST-1 (AQUA SCHWARZ GMBH, 37081 Göttingen, Germany), as well as a suspension of the starter food ST-1 and fine milled brine shrimp nauplii (2 – 8 times daily). 1 day after start of feeding brine shrimp nauplii (48 h old) were fed until the end of the test (4 – 7 times daily).


Light intensity (target) and photoperiod

300 ± 150 Lux
A daily 16 / 8 h photoperiod (light / dark) was maintained throughout exposure.
Reference substance (positive control):
no
Remarks:
No reference item is recommended for this test according to the guideline.
Key result
Duration:
6 d
Dose descriptor:
NOEC
Effect conc.:
>= 13.9 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
number hatched
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 13.9 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
length
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 13.9 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
weight
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 13.9 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Remarks:
Post-hatch survival
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 13.9 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Remarks:
Overall survival
Key result
Duration:
6 d
Dose descriptor:
NOEC
Effect conc.:
>= 28 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
number hatched
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 28 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
length
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 28 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
weight
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 28 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
mortality
Remarks:
Post-hatch survival
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 28 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
mortality
Remarks:
Total survival
Reported statistics and error estimates:
Statistical analysis (NOEC) of hatching success (study days 5 & 6)

Comparison between Control and Solvent control for Hatching Success at Study Day 5 (PHD 0)

Shapiro-Wilk´s Test on Normal Distribution

Treatment [µg/L] Mean s n
Control 1.35 0.000 4
Solvent 1.51 0.113 4

Results:
Number of residuals = 7; Shapiro-Wilk´s W = 0.968; p(W) = 0.703; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Normality check was passed (Shapiro-Wilk´s; p > 0.01).
The F test revealed significantly different variances (p <= 0.01).


STUDENT-t test for Homogeneous Variances

Treatm. [µg/L] Mean s df %MDD t p(t) Sign. p(F)
Control 1.35 0.000
Solvent 1.51 0.113 6 10.3 3.00 0.024 + n.d.
+: significant; -: non-significant

Biological Results


 


Egg Fertilization Rate


The mean egg fertilization rate determined on study day 0 (start of the exposure) was 92%.
Eggs were fully covered with the respective test solutions during fertilization check.


 


Hatch and Definition of Post Hatch Day 0


Hatch began on study day 2 in the test concentration of 28.0 µg/L. The hatch of larvae in other test concentrations and control groups started on study day 3. The hatch of larvae was completed until study day 6. Study day 5 was determined to be post hatch day 0 (PHD 0) with a hatching rate of 95% in the control and 99% in the solvent control (see Table 4).


Statistical procedures were applied for the total number of test organisms that have hatched on study days 5 and 6.
The William’s multiple t-test procedure (alpha = 0.05) for hatch data after 5 and 6 days a significance level of 0.05 was applied. No statistically significant differences were found between the pooled controls and the nominal test concentrations of 7.00 to 28.0 µg/L.
The NOEC and the LOEC (based on nominal test item concentrations) for this endpoint were determined to be ≥ 28.0 and > 28.0 µg/L, corresponding to the arithmetric mean measured concentrations of ≥ 13.9 and > 13.9 µg/L of the dispersed fraction of the test item, respectively.


 


Swim-up


The swim-up period of the control groups and the nominal test concentrations of 7.00 to 28.0 µg/L was observed from study day 5 to 6. First swim-up of larvae was observed on study day 5 in all test groups. For details see Table 5. No statistical analysis of swim-up data was carried out.


 


Fry Survival (Post-Hatch Survival)


The post-hatch survival in the control replicates met the validity criteria of the guideline (required: ≥ 75%). The fry survival (post-hatch survival) at the end of the study was 79% in the control and 84% in the solvent control, thus fully meeting the validity criteria of the guideline given in part 8. No concentration-related decrease of the post-hatch survival was detected with increasing test concentrations. The post-hatch survival data of the test concentrations are given in Table 6.
Dunnett’s multiple t-test was performed on post-hatch survival data on study day 35 (PHD 30). No statistically significant differences were found between the pooled control groups and the nominal test item concentrations of 7.00 to 28.0 µg/L.
The NOEC and the LOEC for this endpoint were ≥ 28.0 µg/L and > 28.0 µg/L (nominal concentrations), corresponding to the arithmetric mean measured concentrations of ≥ 13.9 and > 13.9 µg/L of the dispersed fraction of the test item, respectively.


 


Overall Survival


The cumulative mortality at the end of the exposure, related to the number of eggs introduced on day 0, was 24% for the control and 17% for the solvent control. No concentration-related decrease of the overall survival (increase of overall mortality) was detected in the test item concentrations (see Table 7).
The Dunnett’s multiple t-test was performed for statistical analysis of overall survival data on study day 35 (PHD 30). No statistically significant differences were found between the pooled control groups and the nominal test item concentrations of 7.00 to 28.0 µg/L.
The NOEC and the LOEC for this endpoint were ≥ 28.0 µg/L and > 28.0 µg/L (nominal concentrations), corresponding to the arithmetric mean measured concentrations of ≥ 13.9 and > 13.9 µg/L of the dispersed fraction of the test item, respectively.


 


Morphological and Behavioral Effects


Observation of abnormal appearance and behavior of hatched larvae were carried out daily until the end of the exposure. No morphological and no biological significant behavioral effects were observed in the control, solvent control and in the nominal test concentrations of 7.00 to 28.0 µg/L. The observed behavioural effects were very isolated events of single larvae during exposure.
On study day 8 and 24 one larvae of the control group showed an unusual long arresting on the ground and quiescence.
On study day 11 and 12 one larvae of the solvent control group showed tumbling. On study days 21 to 24 one to two larvae showed an unusual long arresting on the ground and quiescence, respectively.
On study day 6 and 7 two larvae of the lowest test concentration (7.00 µg/L) showed an unusual long arresting on the ground. Tumbling and a slowed escape reflex was also observed for these larvae on study days 7 to 10, respectively.
On study day 24 one larvae on the middle concentration (14.0 µg/L) group showed an unusual long arresting on the ground and quiescence.
On study day 11, 22 to 24 one to two larvae of the highest concentration (28.0 µg/L) group showed an unusual long arresting on the ground and quiescence.


 


Fry Growth


Fry growth, expressed as length and wet weight measurements, was measured on study day 35 (PHD 30) from all survivors (see Table 8 and Table 9). For the individual length data, refer to Table 10 and Table 11. Pooled wet weights based on replicate means are given in Table 12.
The Dunnett’s multiple t-test showed no statistically significant differences for the surviving fish of the nominal test concentrations of 7.00 to 28.0 µg/L, respectively, for both growth parameters (mean total length and fresh weight). Therefore, the NOEC and the LOEC (nominal test item concentrations) for the growth parameter total length were determined to be ≥ 28.0 and > 28.0 µg/L, corresponding to the arithmetric mean measured concentrations of ≥ 13.9 and > 13.9 µg/L of the dispersed fraction of the test item, respectively.


 


Biomass Loading


The biomass-loading factor for the study was determined from the fresh weights of the control and solvent control fish at the end of the exposure (see Table 12).


The maximum biomass at the end of the exposure was determined in replicate 1 of the solvent control group: 711.0 mg total fish weight. The maximum biomass loading based on the 7.5 liter volume of a single growth chamber was 94.8 mg/L.


Maximum loading rate:  biomass / volume of test solution = 711.0 mg / 7.5 L = 94.8 mg/L


The biomass loading rate based upon a flow of 75 liters per day through each single test aquaria was 9.48 mg per liter and day.


Maximum loading rate per day:  biomass / volume of test solution per day = 711.0 mg / 75 L = 9.48 mg/L per day. 


These loadings were well within the requirements to ensure adequate dissolved oxygen levels and to avoid crowding of the fish.


Reported statistics


Statistical Analysis of Hatching Success


Statistical Significance (NOEC) – Hatching Success (Study Days 5 and 6)


Comparison between Control and Solvent control for Hatching Success at Study Day 5 (PHD 0)


Shapiro-Wilk´s Test on Normal Distribution


Shapiro-Wilk´s Test on Normal Distribution with hatchability at 5 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.
Treatm. [µg/L] Mean s n
Control    1.35     0.000    4
Solvent    1.51    0.113     4
Results:
Number of residuals = 7; Shapiro-Wilk´s W = 0.968; p(W) = 0.703; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.


Normality check was passed (Shapiro-Wilk´s; p > 0.01).
The F test revealed siginificantly different variances (p <= 0.01).


STUDENT-t test for Homogeneous Variances


STUDENT-t test for Homogeneous Variances with hatchability at 5 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0.010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt, each).


 


     Treatm. [µg/L]           Mean                  s      df        %MDD                   t                       p(t)           Sign.             p(F)


                 Control             1.35           0.000                                                                                                                   


                 Solvent             1.51           0.113       6             10.3             3.00                    0.024                  +              n.d.


+: significant; -: non-significant


 


A significant difference between Control and each treatment was shown. The effect was considered not to be biological. Therefore, control and solvent control samples were pooled.


Comparison between Control and Solvent control for Hatching Success at Study Day 6


Shapiro-Wilk´s Test on Normal Distribution


Shapiro-Wilk´s Test on Normal Distribution with hatchability at 6 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.


     Treatm. [µg/L]       Mean                s       n


                 Control         1.40         0.113       4


                 Solvent         1.51         0.113       4


Results:


Number of residuals = 6; Shapiro-Wilk´s W = 0.954; p(W) = 0.629; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.


Normality check passed (Shapiro-Wilk´s; p > 0.01).


Variance homogeneity check (F-test) passed (p > 0.01).


STUDENT-t test for Homogeneous Variances


STUDENT-t test for Homogeneous Variances with hatchability at 6 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0,010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt, each).


     Treatm. [µg/L]           Mean                  s      df        %MDD                   t                       p(t)           Sign.             p(F)


                 Control             1.40           0.113                                                                                                                   


                 Solvent             1.51           0.113       6             13.9             1.41                    0.207                   -           1.000


+: significant; -: non-significant


There is no statistically significant difference between control and solvent.


Threshold concentrations (NOEC) for Hatching Success at Study Day 5 (PHD 0)


Shapiro-Wilk´s Test on Normal Distribution


Shapiro-Wilk´s Test on Normal Distribution with hatchability at 5 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.


     Treatm. [µg/L]       Mean                s       n


     Pooled Control         1.43          0.117        8


                    7.000         1.51         0.113       4


                  14.000         1.49         0.161       4


                  28.000         1.40         0.113       4


Results:


Number of residuals = 8; Shapiro-Wilk´s W = 0.946; p(W) = 0.613; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.


Levene´s Test on Variance Homogeneity (with Residuals)


Levene´s Test on Variance Homogeneity (with Residuals) with hatchability at 5 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance


                 Source                      SS                         df                     MSS                           F                    p(F)


            Treatment                0.0039                           3                  0.0013                    0.465                  0.711


             Residuals                0.0444                         16                  0.0028                                                        


                     Total                  0.048                         19                                                                                      


The Levene test indicates variance homogeneity (p > 0.010).


Variance homogeneity check was passed (p > 0.01).


Normal-distribution and variance-homogeneity requirements are fulfilled and so the user-selected multiple test was performed.


To justify the use of Williams test at first a trend analysis by contrasts was performed.


Trend analysis by Contrasts (Monotonicity of Concentration/Response)


Trend analysis by contrasts (monotonicity of concentration/response) with hatchability at 5 d:  Psi: sum of means weighted by contrasts; s(psi): standard error of psi; df: degrees of freedom;  t: t-statistic; p(t): probability that the trend is due to chance (Ho: Slope = 0). Hypothesis of monotonicity is accepted if at least the linear contrast is significant.


               Trend                  Psi               s(psi)                     df                       t                 p(t)


               Linear            0,1086              0,2457                    16                0,442              0,332


          Quadratic            0,1733              0,1168                    16                1,484              0,079


The linear trend was not significant (p > 0,05) The quadratic trend was not significant (p > 0,05)


The analysis of contrasts did not reveal a linear trend, nonetheless the user-selected Williams test was performed.


Williams Multiple Sequential t-test Procedure


Comparison of treatments with "Pooled Control" by the t test procedure after Williams with hatchability at 5 d: Significance was Alpha = 0.050, one-sided smaller; Mean: arithmetic mean; n: sample size; s: standard deviation; LhM: max. likelihood mean; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; 't*: critical t for Ho: µ1 = µ2 = ... = µk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments). Note that the step-down test terminates after the first non-significant treatment is encountered


     Treatm. [µg/L]       Mean                s      df         LhM        %MDD                    t                          t*       Sign.


     Pooled Control         1.43       0.1248                                                                                                              


                    7.000         1.51       0.1248     16         1.51              -9.3               1.11                     -1.75               -


                  14.000         1.49       0.1248     16         1.49              -9.7               0.79                     -1.82               -


                  28.000         1.40       0.1248     16         1.40              -9.8             -0.37                     -1.84               -


+: significant; -: non-significant 


The NOEC appears to be higher than or equal 28.0 µg/L.


Threshold concentrations (NOEC) for Hatching Success at Study Day 6 


Shapiro-Wilk´s Test on Normal Distribution


Shapiro-Wilk´s Test on Normal Distribution with hatchability at 6 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) was accepted.


     Treatm. [µg/L]       Mean                s       n


     Pooled Control         1.46         0.121       8


                    7.000         1.51         0.113       4


                  14.000         1.49         0.161       4


                  28.000         1.40         0.113       4


Results:


Number of residuals = 8; Shapiro-Wilk´s W = 0.948; p(W) = 0.620; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.


 


Levene´s Test on Variance Homogeneity (with Residuals)


Levene´s Test on Variance Homogeneity (with Residuals) with hatchability at 6 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance


                 Source                      SS                         df                     MSS                           F                    p(F)


            Treatment                0.0047                           3                  0.0016                    0.655                  0.591


             Residuals                0.0385                         16                  0.0024                                                        


                     Total                  0.043                         19                                                                                      


The Levene test indicated variance homogeneity (p > 0.010).


Variance homogeneity check was passed (p > 0.01).


Normal-distribution and variance-homogeneity requirements were fulfilled, and so parametric multiple test was appropriate.


To justify the use of Williams test at first a trend analysis by contrasts was performed.


 


Trend analysis by Contrasts (Monotonicity of Concentration/Response)


Trend analysis by contrasts (monotonicity of concentration/response) with hatchability at 6 d:  Psi: sum of means weighted by contrasts; s(psi): standard error of psi; df: degrees of freedom;  t: t-statistic; p(t): probability that the trend is due to chance (Ho: Slope = 0). Hypothesis of monotonicity is accepted if at least the linear contrast is significant.


                   Trend                      Psi                   s(psi)                         df                            t                     p(t)


                   Linear                0.1932                  0.2488                         16                    0.776                  0.224


             Quadratic                0.1451                  0.1182                         16                    1.227                  0.119


The linear trend is not significant (p > 0.05) The quadratic trend is not significant (p > 0.05)


The analysis of contrasts did not reveal a linear trend, thus the selected Williams test was replaced by Dunnett test.


Dunnett`s Multiple t-test Procedure


Dunnett´s multiple t-test procedure with hatchability at 6 d: Comparison of treatments with "Pooled Control". Significance was Alpha = 0,050, one-sided smaller (multiple level); Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; t*: critical t for Ho: µ1 = µ2 = ... = µk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).


     Treatm. [µg/L]       Mean                s      df        %MDD                   t                   t*                    Sign.


     Pooled Control         1.46       0.1264                                                                       


                    7.000         1.51       0.1264     16            -12.1             0.73             -2.27 -


                  14.000         1.49       0.1264     16            -12.1             0.42             -2.27 -


                  28.000         1.40       0.1264     16            -12.1            -0.73             -2.27 -


+: significant; -: non-significant


The NOEC appears to be higher than or equal 28.0 µg/L.


 


Statistical Analysis of Post-Hatch Survival


Statistical Significance (NOEC) – Post-Hatch Survival on Study Day 35 (PHD 30)


Comparison between Control and Solvent control for Post-hatch Survival at Study Day 35


Shapiro-Wilk´s Test on Normal Distribution


Shapiro-Wilk´s Test on Normal Distribution with post-hatch survival at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.


     Treatm. [µg/L]       Mean                s       n


                 Control         1.11         0.177       4


                 Solvent         1.17         0.145       4


Results:


Number of residuals = 18; Shapiro-Wilk´s W = 0.958; p(W) = 0.557; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.


Normality check was passed (Shapiro-Wilk´s; p > 0.01).


Variance homogeneity ckeck (F-test) was passed (p > 0.01).


STUDENT-t test for Homogeneous Variances


STUDENT-t test for Homogeneous Variances with post-hatch survival at 35 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0,010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt, each).


     Treatm. [µg/L]           Mean                  s      df        %MDD                   t                       p(t)           Sign.             p(F)


                 Control             1.11           0.177                                                                                                                   


                 Solvent             1.17           0.145       6             25.1             0.48                    0.649                   -           0.747


+: significant; -: non-significant


There was no statistically significant difference between control and solvent.


Threshold concentrations (NOEC) for Post-Hatch Survival


Shapiro-Wilk´s Test on Normal Distribution


Shapiro-Wilk´s Test on Normal Distribution with post-hatch survival at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level. the normality hypothesis(Ho) is accepted.


     Treatm. [µg/L]       Mean                s       n


        Pooled Control         1.14         0.153       8


                    7.000         1.07         0.126       4


                  14.000         1.19         0.043       4


                   28.000        1.25         0.217       4


Results:


Number of residuals = 18; Shapiro-Wilk´s W = 0.967; p(W) = 0.734; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.


 


Levene´s Test on Variance Homogeneity (with Residuals)


Levene´s Test on Variance Homogeneity (with Residuals) with post-hatch survival at 35 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance


                 Source                      SS                         df                     MSS                           F                    p(F)


            Treatment                0.0353                           3                  0.0118                    1.688                  0.210


             Residuals                0.1115                         16                  0.0070                                                        


                     Total                  0.147                         19                                                                                      


The Levene test indicates variance homogeneity (p > 0.010).


Variance homogeneity check was passed (p > 0.01).


Normal-distribution and variance-homogeneity requirements are fulfilled.


A parametric multiple test was suitable.


To justify the use of Williams test at first a trend analysis by contrasts is performed.


 


Trend analysis by Contrasts (Monotonicity of Concentration/Response)


Trend analysis by contrasts (monotonicity of concentration/response) with post-hatch survival at 35 d:  Psi: sum of means weighted by contrasts; s(psi): standard error of psi; df: degrees of freedom;  t: t-statistic; p(t): probability that the trend is due to chance (Ho: Slope = 0). Hypothesis of monotonicity is accepted if at least the linear contrast is significant.


                   Trend                      Psi                   s(psi)                         df                            t                     p(t)


                   Linear                0.4495                  0.2941                         16                    1.528                  0.073


             Quadratic                0.1388                  0.1398                         16                    0.993                  0.168


The linear trend was not significant (p > 0.05) The quadratic trend is not significant (p > 0.05)


The analysis of contrasts did not reveal a linear trend, thus the selected Williams test was replaced by Dunnett test.


 


Dunnett`s Multiple t-test Procedure


Dunnett´s multiple t-test procedure with post-hatch survival at 35 d: Comparison of treatments with "Pooled Control". Significance was Alpha = 0.050, one-sided smaller (multiple level); Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; t*: critical t for Ho: µ1 = µ2 = ... = µk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).


     Treatm. [µg/L]       Mean                s      df        %MDD                   t                   t*                    Sign.


       Pooled Control         1.14       0.1494                                                                       


                    7.000         1.07       0.1494     16            -18.2            -0.82             -2.27 -


                  14.000         1.19       0.1494     16            -18.2             0.50             -2.27 -


                  28.000         1.25       0.1494     16            -18.2             1.20             -2.27 -


+: significant; -: non-significant


The NOEC appears to be higher than or equal 28.0 µg/L.


 


Statistical Analysis of Overall Survival


Statistical Significance (NOEC) – Overall Survival Study Day 35 (PHD 30)
Comparison between Control and Solvent control for Overall Survival


Shapiro-Wilk´s Test on Normal Distribution


Shapiro-Wilk´s Test on Normal Distribution with surival at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.


     Treatm. [µg/L]       Mean                s       n


                 Control         1.07         0.161       4


                 Solvent         1.15         0.148       4


Results:


Number of residuals = 18; Shapiro-Wilk´s W = 0.965; p(W) = 0.700; p(W) is greater than the selected significance level of 0.010; thus treatment data did not significantly deviate from normal distribution.


Normality check was passed (Shapiro-Wilk´s; p > 0.01).


Variance homogeneity check (F-test) was passed (p > 0.01).


STUDENT-t test for Homogeneous Variances


STUDENT-t test for Homogeneous Variances with surival at 35 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0,010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt, each).


     Treatm. [µg/L]           Mean                  s      df        %MDD                   t                       p(t)           Sign.             p(F)


                 Control             1.07           0.161                                                                                                                   


                 Solvent             1.15           0.148       6             24.9             0.75                    0.482                   -           0.894


+: significant; -: non-significant


There was no statistically significant difference between control and solvent.


Threshold concentrations (NOEC) for Overall Survival


Shapiro-Wilk´s Test on Normal Distribution


Shapiro-Wilk´s Test on Normal Distribution with surival at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.


     Treatm. [µg/L]       Mean                s       n


        Pooled Control         1.11           0.150     8


                    7.000         1.06           0.136     4


                  14.000         1.16           0.084     4


                  28.000         1.17           0.129     4


Results:


Number of residuals = 16; Shapiro-Wilk´s W = 0.960; p(W) = 0.660; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.


 


Levene´s Test on Variance Homogeneity (with Residuals)


Levene´s Test on Variance Homogeneity (with Residuals) with surival at 35 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance


                 Source                      SS                         df                     MSS                           F                    p(F)


            Treatment                0.0059                           3                  0.0020                    0.276                  0.842


             Residuals                0.1151                         16                  0.0072                                                        


                     Total                  0.121                         19                                                                                      


The Levene test indicates variance homogeneity (p > 0.010).


Variance homogeneity check was passed (p > 0.01).


Normal-distribution and variance-homogeneity requirements are fulfilled.


A parametric multiple test is advisable.


To justify the use of Williams test at first a trend analysis by contrasts is performed.


 


Trend analysis by Contrasts (Monotonicity of Concentration/Response)


Trend analysis by contrasts (monotonicity of concentration/response) with surival at 35 d:  Psi: sum of means weighted by contrasts; s(psi): standard error of psi; df: degrees of freedom;  t: t-statistic; p(t): probability that the trend is due to chance (Ho: Slope = 0). Hypothesis of monotonicity is accepted if at least the linear contrast is significant.


                   Trend                      Psi                   s(psi)                         df                            t                     p(t)


                   Linear                0.2693                  0.2618                         16                    1.029                  0.160


             Quadratic                0.0645                  0.1244                         16                    0.518                  0.306


The linear trend is not significant (p > 0.05) The quadratic trend is not significant (p > 0.05)


The analysis of contrasts did not reveal a linear trend. thus the selected Williams test was replaced by Dunnett test.


Dunnett`s Multiple t-test Procedure


Dunnett´s multiple t-test procedure with surival at 35 d: Comparison of treatments with "Pooled Control". Significance was Alpha = 0.050. one-sided smaller (multiple level); Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; t*: critical t for Ho: µ1 = µ2 = ... = µk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).


     Treatm. [µg/L]       Mean                s      df        %MDD                   t                   t*                    Sign.


     Pooled Control         1.11       0.1330                                                                       


                    7.000         1.06       0.1330     16            -16.6            -0.70             -2.27 -


                  14.000         1.16       0.1330     16            -16.6             0.58             -2.27 -


                  28.000         1.17       0.1330     16            -16.6             0.68             -2.27 -


+: significant; -: non-significant


The NOEC appears to be higher than or equal 28.0 µg/L.


 


Statistical Analysis of Fry Growth – Fresh Weight


Statistical Significance (NOEC) – Fry Growth: Fresh Weight on Study Day 35 (PHD 30)


Comparison between Control and Solvent control for Fresh Weight


Shapiro-Wilk´s Test on Normal Distribution


Shapiro-Wilk´s Test on Normal Distribution with fresh weight at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.


     Treatm. [µg/L]       Mean                s       n


                 Control     39.750       6.7501       4


                 Solvent     38.025       3.8257       4


Results:


Number of residuals = 19; Shapiro-Wilk´s W = 0.941; p(W) = 0.278; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.


Normality check was passed (Shapiro-Wilk´s; p > 0.01)


Variance homogeneity ckeck (F-test) was passed (p > 0.01).


STUDENT-t test for Homogeneous Variances


STUDENT-t test for Homogeneous Variances with fresh weight at 35 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050. two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0.010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt. each).


     Treatm. [µg/L]           Mean                  s      df        %MDD                   t                       p(t)           Sign.             p(F)


                 Control         39.750         6.7501                                                                                                                   


                 Solvent         38.025         3.8257       6             23.9            -0.44                    0.672                   -           0.376


+: significant; -: non-significant


There is no statistically significant difference between control and solvent.


Threshold concentrations (NOEC) for Fresh Weight


Shapiro-Wilk´s Test on Normal Distribution


Shapiro-Wilk´s Test on Normal Distribution with fresh weight at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.


     Treatm. [µg/L]       Mean                s       n


     Pooled Control     38.888       5.1623       8


                    7.000     41.575       3.4023       4


                  14.000     34.425       2.9239       4


                  28.000     34.250       2.0273       4


Results:


Number of residuals = 19; Shapiro-Wilk´s W = 0.930; p(W) = 0.173; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.


 


Levene´s Test on Variance Homogeneity (with Residuals)


Levene´s Test on Variance Homogeneity (with Residuals) with fresh weight at 35 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance


                 Source                      SS                         df                     MSS                           F                    p(F)


            Treatment            13.72290                           3                4.57430                    0.737                  0.545


             Residuals            99.32552                         16                6.20784                                                        


                     Total            113.0484                         19                                                                                      


The Levene test indicates variance homogeneity (p > 0.010).


Variance homogeneity check was passed (p > 0.01).


Normal-distribution and variance-homogeneity requirements are fulfilled.


The user-selected multiple test is performed, according to the statistical guidance (annex5) of the test guideline OECD 210.


Dunnett`s Multiple t-test Procedure


Dunnett´s multiple t-test procedure with fresh weight at 35 d: Comparison of treatments with "Pooled Control". Significance was Alpha = 0.050. one-sided smaller (multiple level); Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; t*: critical t for Ho: µ1 = µ2 = ... = µk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).


     Treatm. [µg/L]       Mean                s      df        %MDD                   t                   t*                    Sign.


     Pooled Control     38.888     4.02534                                                                       


                    7.000     41.575     4.02534     16            -14.4             1.09             -2.27 -


                  14.000     34.425     4.02534     16            -14.4            -1.81             -2.27 -


                  28.000     34.250     4.02534     16            -14.4            -1.88             -2.27 -


+: significant; -: non-significant


The NOEC appears to be higher than or equal 28.0 µg/L.


 


Statistical Analysis of Fry Growth – Mean Total Length


Statistical Significance (NOEC) – Fry Growth: Mean Total Length on Study Day 35 (PHD 30)
Comparison between Control and Solvent control for Mean Total Length


Shapiro-Wilk´s Test on Normal Distribution


Shapiro-Wilk´s Test on Normal Distribution with length at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.


     Treatm. [µg/L]       Mean                s       n


                 Control       15.85         0.968       4


                 Solvent       15.40         0.762       4


Results:


Number of residuals = 19; Shapiro-Wilk´s W = 0.931; p(W) = 0.184; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.


Normality check was passed (Shapiro-Wilk´s; p > 0.01).


Variance homogeneity ckeck (F-test) was passed (p > 0.01).


STUDENT-t test for Homogeneous Variances


STUDENT-t test for Homogeneous Variances with length at 35 d: Two-sample comparison of the two controls. Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(t) <= Alpha ; p(F): two-sided probability of F computed by the F-test (Ho: var1 = var2 (homogeneity); p(F) > 0,010 is the criterion of variance homogeneity. (Control(c) and treatment(t) variance was applied: s²(c)/nc + s²(t)/nt, each).


     Treatm. [µg/L]           Mean                  s      df        %MDD                   t                       p(t)           Sign.             p(F)


                 Control           15.85           0.968                                                                                                                   


                 Solvent           15.40           0.762       6               9.5            -0.73                    0.492                   -           0.703


+: significant; -: non-significant


There is no statistically significant difference between control and solvent.


 


Threshold concentrations (NOEC) for Mean Total Length


Shapiro-Wilk´s Test on Normal Distribution


Shapiro-Wilk´s Test on Normal Distribution with length at 35 d: Mean: arithmetic mean; n: sample size; p(ShapiroWilk´s W): probability of the W statistic (i.e. that the observed deviations from the normal distributions are dues to chance). In case p(ShapiroWilk´s W) is greater than the chosen significance level, the normality hypothesis(Ho) is accepted.


     Treatm. [µg/L]       Mean                s       n


     Pooled Control       15.62         0.841       8


                    7.000       15.82         0.741       4


                  14.000       15.40         0.548       4


                  28.000       15.17         0.403       4


Results:


Number of residuals = 18; Shapiro-Wilk´s W = 0.963; p(W) = 0.663; p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.


 


Levene´s Test on Variance Homogeneity (with Residuals)


Levene´s Test on Variance Homogeneity (with Residuals) with length at 35 d: Source: source of variance; SS: sum of squares; df: degrees of freedom; MSS: mean sum of squares; F: test statistic: p: probability that the variance explained by the treatment is due to chance


                 Source                      SS                         df                     MSS                           F                    p(F)


            Treatment                0.2857                           3                  0.0952                    0.585                  0.633


             Residuals                2.6037                         16                  0.1627                                                        


                     Total                  2.889                         19                                                                                      


The Levene test indicates variance homogeneity (p > 0.01).


Variance homogeneity check was passed (p > 0.01)


Normal-distribution and variance-homogeneity requirements are fulfilled.


A parametric multiple test is advisable.


To justify the use of Williams test at first a trend analysis by contrasts is performed.


 


Trend analysis by Contrasts (Monotonicity of Concentration/Response)


Trend analysis by contrasts (monotonicity of concentration/response) with length at 35 d:  Psi: sum of means weighted by contrasts; s(psi): standard error of psi; df: degrees of freedom;  t: t-statistic; p(t): probability that the trend is due to chance (Ho: Slope = 0). Hypothesis of monotonicity is accepted if at least the linear contrast is significant.


                   Trend                      Psi                   s(psi)                         df                            t                     p(t)


                   Linear                1.7750                  1.3911                         16                    1.276                  0.110


             Quadratic                0.4250                  0.6610                         16                    0.643                  0.265


The linear trend is not significant (p > 0.05) The quadratic trend is not significant (p > 0.05)


The analysis of contrasts did not reveal a linear trend, thus the selected Williams test was replaced by Dunnett test.


Dunnett`s Multiple t-test Procedure


Dunnett´s multiple t-test procedure with length at 35 d: Comparison of treatments with "Pooled Control". Significance was Alpha = 0.050, one-sided smaller (multiple level); Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Pooled Control (in percent of Pooled Control); t: sample t; t*: critical t for Ho: µ1 = µ2 = ... = µk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).


     Treatm. [µg/L]       Mean                s      df        %MDD                   t                   t*                    Sign.


     Pooled Control       15.62       0.7067                                                                       


                    7.000       15.82       0.7067     16              -6.3             0.46             -2.27 -


                  14.000       15.40       0.7067     16              -6.3            -0.52             -2.27 -


                  28.000       15.17       0.7067     16              -6.3            -1.04             -2.27 -


+: significant; -: non-significant


The NOEC appears to be higher than or equal 28.0 µg/L.


 


 


 

Validity criteria fulfilled:
yes
Remarks:
Dissolved oxygen saturation - between 84 and 100%. Water temperature was in the recommended range. Hatching success was 96% in the control and 99% in the solvent control groups. Post-hatch survival was 79% in control and 84% in solvent control groups.
Conclusions:
1, 2, 4-Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS-No. 90218-76-1) caused no significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations of 7.00, 14.0 and 28.0 µg/L.

For the parameter hatching success, the nominal NOEC was determined to be ≥ 28.0 µg/L. Corresponding to the arithmetric mean measured concentration NOEC of ≥ 13.9 µg/L of the dispersed fraction of the test item.

For the parameters post hatch survival and overall survival, the nominal NOECs were determined to be ≥ 28.0 µg/L, corresponding to the arithmetric mean measured NOEC of ≥ 13.9 µg/L of the dispersed fraction of the test item.

For the parameter fry growth (expressed as length and fresh weight) the nominal NOECs were ≥ 28.0 µg/L (for both parameters). Corresponding to the arithmetric mean measured concentration NOECs of ≥ 13.9 µg/L of the dispersed fraction of the test item.

All effect values are given based on the nominal test item concentrations and the arithmetric mean measured concentrations of the dispersed fraction of the test item 1, 2, 4-Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS-No. 90218-76-1).
Executive summary:

The effects of the test item 1, 2, 4 Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS-No. 90218-76-1) (Batch-No. 05804/MA) on the early-life stage of fish (Danio rerio / Zebrafish) were determined according to OECD Guideline 210. 


Stock solutions in ethanol with nominal concentrations of 280, 560 and 1120 mg/L were prepared at intervals of 7 days and continuously dosed in a flow-through system. Based on the results of a range finding test, a dose-response test was conducted with nominal test item concentrations 7.00, 14.0 and 28.0 µg/L, corresponding to the arithmetric mean measured concentrations 4.72 - 8.45 - 13.9 µg/L of the dispersed fraction of the test item.


The test lasted 35 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each).


Water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.


On study day 5, 95% of the control and 99% the solvent control larvae had hatched. Therefore, study day 5 was defined as post hatch day 0 (= PHD 0).


The following toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, post-hatch survival and overall survival.


Concentrations of the test item and controls were determined during exposure on day 0, day 7, day 14, day 21 and day 28 for all tested concentration levels and for three constituents C8, C8, C10-BTE, C8, C10, C10 BTE and C10, C10, C10-BTE via LC MS/MS.


1, 2, 4-Benzenetricarboxylic acid, mixed decyl and octyl triesters (CAS-No. 90218-76-1) caused no significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations of 7.00, 14.0 and 28.0 µg/L.


For all parameters measured, the nominal NOEC was determined to be 28.0 µg/L, which was the highest dose tested. Corresponding to the arithmetric mean measured concentration NOEC of 13.9 µg/L of the dispersed fraction of the test item. This represents the maximum achievable concentration due to the low solubility of the test item. As such, there were no significant effects at the limit of solubility. The NOECs have been reported for completeness but are not appropriate for classification or risk assessment.

Description of key information

In one OECD 210 study, for parameters: hatching success, survival and growth, the nominal NOEC was determined to be 28.0 µg/L, corresponding to the arithmetric mean measured concentration NOEC of 13.9 µg/L of the dispersed fraction of the test item. This represents the highest achievable concentration due to the low solubility of the test item. There were no significant effects at the limit of solubility and therefore the NOEC values are not appropriate for classification and risk assessment.

Key value for chemical safety assessment

Additional information