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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:
From November 30, 2021 to March 23, 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:
Adopted: 26 July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Prior to the proposed test start, the flow through system was equilibrated and samples from all test vessels were taken for chemical analysis. At test start, test medium from all test vessels was sampled and measured.
In the following, samples from two replicates of each exposure group, i.e. control, solvent control and treatment, were taken once weekly. The replicates were sampled alternately, i.e. replicate A and C in week 1, replicate B and D in week 2, etc.
Samples were immediately transferred to the chemical analysis laboratory and measured. In case that test samples could not be analysed within 24 hours, storage stability experiments were included.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: flow-through test conditions
- Dilution water: Purified drinking water, sourced from the Schmallenberg district water production plants, mostly fed by small springs and percolation, was used. The purification included filtration with activated charcoal and aeration. To avoid copper contamination, plastic water pipes are used for the test facilities. The water was aerated to the point of oxygen saturation.
- Controls: a water control and a solvent control were included
- Chemical name of vehicle: Acetone, used at 12 µL/L
- Test concentration: Based on the results of a non-GLP pretest, the following test concentrations were applied in definitive study: 500, 158, 50, 15.8 and 5.0 µg/L (spacing factor 3.16)
- Other relevant information: to minimize impacts of bacterial growth due to the application of solvent, the vessels were cleaned in regular intervals. Furthermore, the flexible tubes serving the test solutions were rinsed or exchanged at least once a week
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish (Danio rerio, Teleostei, Cyprinidae)
- Source: Fertilised eggs for the study were obtained from individuals reared at the test facility.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
Eggs were collected in a glass spawning-tray, which was placed at the bottom of the holding vessels. The tray was covered with a stainless steel lattice to prevent adult fish from predating the eggs. An artificial substrate was attached to the lattice to stimulate spawning into the tray.
The turn on of lighting (one neon lamp per vessel, light intensity approximately 1000 lux, measured 5 cm above the water surface in the middle of the test vessel) induced mating and spawning of fish.
The collected eggs were transferred from the spawning-tray into a sieve, rinsed with clean water in order to remove any debris and then put into glass dishes. Fertilized eggs (microscopic determination of >four cell stage, i.e. early blastula stage) were then transferred by means of a widened and de-burred pipette tip into the test chambers. Time from spawning until transferring into the test solutions was kept as short as possible.

POST-HATCH FEEDING
At test start, 20 fertilized and randomized eggs were placed in suitable fry cages placed in every test beaker. Each aquarium was equipped with one cage. 80 eggs (20 eggs to each of the four replicates) were used for the control, the solvent control and the test concentration each.
When hatch was finished, e.g. from day 5 dpf on (dpf = days post fertilization), larvae were fed at least twice daily with ground breeding food (TetraMin Baby, Tetra Werke, Melle, Germany) and liquid rearing feed (Nobil fluid, JBL, Neuhofen, Germany). From day 14 (dpf) on, brine shrimp nauplii (Artemia salina) were added twice daily. From day 21 onwards, breeding food was exchanged by ground TetraMin flake food (Tetra Werke, Melle, Germany), ad libitum. Uneaten food and faeces were removed from the fry cages and the test aquaria at regular intervals.
Test type:
flow-through
Water media type:
freshwater
Remarks:
Purified drinking water water was sourced from the Schmallenberg district water production plants. The purification included filtration with activated charcoal and aeration.
Limit test:
no
Total exposure duration:
35 d
Hardness:
1.0 - 1.1 mmol/L
Test temperature:
26.0 °C to 26.6 °C
pH:
between 7.50 and 8.21
Dissolved oxygen:
between 68.0 and 102.0% of saturation
Conductivity:
251 - 257 µS/cm
Nominal and measured concentrations:
Nominal concentrations: 5.0, 15.8, 50, 158 and 500 µg/L
Mean concentrations: 6.05, 21.6, 64.5, 153 and 443 µg/L, between 88.6 % and 136 % of the nominal concentration.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass aquaria
- Size, headspace, fill volume: total volume of 6.5 L and approx. 5 L of test medium, the fish density did not exceed 5 g fish/L during the test.
- Fry cage: each test vessel was equipped with a fry cage, a glass cylinders (8 cm diameter, 10 cm height) with a sieve net at the bottom (mesh width of 355 μm).
- Aeration: The holding water was aerated to the point of oxygen saturation before introducing to the test system.
- Type of flow-through: Dilution water was pumped by a water dosage pump (membrane pump, Prominent, Heidelberg, Germany) into a mixing chamber, placed on a magnetic stirrer
- Renewal rate of test solution: a daily exchange rate of ten volumes per vessel and day was adjusted.
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Drinking water purified including filtration with activated charcoal and aeration.
- Total organic carbon: The Non purgeable organic carbon method was used for detection of total organic carbon (TOC): 1.069 - 1.369 mg/L.
- Metals (in µg/L):
Cadmium: 0.004 - 0.008
Chrome: 0.066 - 0.084
Copper: 0.414 - 2.66
Iron: 0.587 - 3.34
Manganese: 0.042 - 1.234
Nickel: 0.351 - 0.922
Lead: 0.009 - 0.131
Zinc: 0.927 - 4.965
- Pesticides: none
- Chlorine: < 0.02 mg/L (i.e. < LOQ)
- Alkalinity: 1.3 - 1.8 mmol/L
- Ca/Mg ratio: 3 to 8
- Intervals of water quality measurement: The water chemistry data were recorded regularly, i.e. each month, in the test facility.

OTHER TEST CONDITIONS
- Adjustment of pH: the pH value was between 7.295 - 7.763 without adjustment
- Photoperiod: 12 hours light / 12 hours dark
- Light intensity: approximately 1000 lux

EFFECT PARAMETERS MEASURED (sorted as followed: Course / Study duration or Fish life [days post fertilisation] / Endpoints
Introduction of 20 eggs per vessel (4 x 20 = 80 eggs per treatment) / 0 / Mortality/survival rate
Begin of hatch / 2 - 3 / Mortality/survival rate, hatching success
End of sac fry stage / 9 - 10 / Mortality / survival rate
Transfer of fish larvae to test vessels / 14 / Mortality
Photo and post hatch survival / 21 / Mortality (survival rate)
Photo, end of the study / 35 / Mortality (survival rate), length, weight

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: nominal 5.0, 50, and 500 µg/L. Two replicates per test concentration plus two control replicates, each containing 20 fertilised eggs, were applied for a duration of 21 days.
- Results used to determine the conditions for the definitive study: No effect on hatch was observed for any treatment level. A effect on post hatch survival, with no larvae surviving was observed at the highest test concentration.

POST-HATCH DETAILS
- Begin of post-hatch period: Hatch was completed at day 5 post fertilisation in all treatments and the controls.
- No. of hatched eggs/treatment released to the test chamber: Hatching success was between 98.8 and 100 % in all treatments and the controls.
Reference substance (positive control):
no
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
64.5 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks:
A statistically significant effect on post hatch survival at study end was detected in the nominal 158 and 500 µg/L treatment groups
Key result
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
78.4 µg/L
95% CI:
11.6 - 108.4
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks:
A statistically significant effect on post hatch survival at study end was detected in the nominal 158 and 500 µg/L treatment groups
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
153 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
weight
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
153 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
length
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
153 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks:
Post hatch survival 21 days post fertilisation was not statistically significant different between the controls and the nominal treatment levels of 5.00, 15.8, 50.0 and 158 µg/L. A statistically significant mortality was observed at nominal 500 µg/L
Duration:
5 d
Dose descriptor:
NOEC
Effect conc.:
> 443 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
time to hatch
Remarks:
all eggs hatched between 3 and 5 days with no difference between any treatment and the controls
Duration:
5 d
Dose descriptor:
NOEC
Effect conc.:
> 443 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
number hatched
Remarks:
Hatching success was 100 % for the controls and all test item concentrations except in the nominal 158 µg/L treatment group (98.8 % hatching success).
Details on results:
Water quality parameters
The temperature was in the range of 26.0 °C to 26.6 °C in all test vessels throughout the in-life phase of the study and did not differ by more than 1.5 °C on successive days and thus was in line with the guideline requirements. The oxygen saturation in all test vessels was between 68.0 and 102.0 %. The pH in the test vessels was between 7.50 and 8.21. All values were within the range stated in the guideline.

Hatching rate
First larvae hatched at day 3 post fertilisation (pf) across all treatment levels. Hatch was completed at day 5 pf in all replicates, with no difference between treatments. Coagulated eggs were not found neither in the controls nor in treatments with the test item.
The mean hatching success was 100 % in control and solvent-control. In treatment with 6.05, 21.6, 64.5, 153 and 443 µg/L, the mean hatching success was assessed to be between 98.8 and 100 %.

Post hatch survival
Survival was recorded at day 21 pf and at test end, i.e. at day 35 pf. Mortality of larvae occurred mainly before 21 dpf, during the phase of transition from yolk sac feeding to external feeding.
At test end, the post hatch survival rate in control and solvent-control reached a mean value of 86.3 and 78.8 % and thus met the validity criterion for survival in controls of ≥ 75 %.
The post hatch survival in treatments was determined to be 83.3, 87.5, 90.0, 36.6 and 5.0 % in the test concentrations of 6.05, 21.6, 64.5, 153 and 443 µg/L (mean measured), respectively. There was a statistically significant decrease of survival in treatment with mean measured 153 µg/L and 443 µg/L compared to the solvent control (Williams Multiple t-test, one-sided smaller, p<0.05).

Size
At test end, larval growth in terms of length and weight were determined. With respect to the test guideline, larvae in controls should reach a mean length of 11 mm. In this study, the mean total lengths were determined to be 18 mm in the control and 16 mm in solvent-control. Thus, the growth performance of the fish larvae was in line with the guideline specifications. The mean total lengths in the treatment groups were determined to be 18, 17, 17, 17 and 14 mm in test concentrations of 6.05, 21.6, 64.5, 153 and 443 µg/L (mean measured), respectively.
Statistical analyses revealed a significant reduction of larval length compared to solvent-control in treatment with 443 µg/L (Williams Multiple t-test; p=0.05; one-sided smaller).
The mean individual dry weights were determined to be 15.7 mg and 6.9 mg in the control and the solvent-control, respectively. The mean individual dry weights in treatments were assessed to be 12.3, 12.4, 11.6, 10.4 and 4.3 at test concentrations of 6.05, 21.6, 64.5, 153 and 443 µg/L (mean measured), respectively.
Statistical analyses revealed a significant reduction compared to solvent control in treatment with 443 µg/L (Williams Multiple t-test; p=0.05; one-sided smaller).

No abnormal appearance or behaviour was reported.
Reported statistics and error estimates:
For each endpoint, the NOEC, LOEC and ECx values with confidence intervals were determined, if possible. For NOEC / LOEC-determination, quantal data were arcsine-transformed prior to analysis. No Observed Effect Concentrations (NOEC) were calculated using ANOVA, followed by appropriate post-hoc tests or respective non-parametric approaches. ECx values were calculated by Logit analysis using linear max. likelihood regression. Prior to use of parametric procedures, results of tests of normality and homogeneity of variance were considered. All statistics were calculated using the program ToxRat Professional 3.3.0.
In accordance to OECD 23 all results were based on the solvent control as relevant control.

Table 1: Biological effects during larval development; hatching success and post hach survival









































































































































 



Nominal concentration [µg/L]



 



control



solvent-control



5.00



15.8



50.0



158



500



 



Mean measured concentration [µg/L]



 



control



solvent-control



6.05



21.6



64.5



153



443



No. of replicates



4



4



4



4



4



4



4



Hatch,
day 5 pf


[%]



Mean



100



100



100



100



100



98.8



100



SD



0.0



0.0



0.0



0.0



0.0



2.5



0.0



RSD



0.0



0.0



0.0



0.0



0.0



2.5



0.0



Post hatch survival,
at 21 dpf [%]



Mean



86.3



78.8



85.0



88.8



91.3



41.6



7.5 1)



SD



8.5



7.5



10.8



6.3



7.5



42.3



15.0



RSD



9.9



9.5



12.7



7.1



8.2



101.8



200.0



Post hatch survival,
at test end [%]



Mean



86.3



78.8



83.8



87.5



90.0



36.6 2)



5.0 2)



SD



8.5



7.5



12.5



6.5



9.1



33.4



10.0



RSD



9.9



9.5



14.9



7.4



10.1



91.2



200.0



1), 2)        Williams test, one-sided smaller, p<0.05 (quantal data arcsine-transformed prior to statistical evaluation), compared to the solvent control


 


 


Table 2: Biological effects during larval development; growth parameters at test end










































































































 



Nominal concentration [µg/L]



 



control



solvent-control



5.00



15.8



50.0



158



500



 



Mean measured concentration [µg/L]



 



control



solvent-control



6.05



21.6



64.5



153



443



No. of replicates



4



4



4



4



4



3



1



Length,
at test end [mm]



Mean



18



16



18



17



17



17



14 3)



SD



0.7



0.3



0.5



0.8



1.1



0.7



-



RSD



3.9



1.6



2.7



4.5



6.6



4.1



-



Single fish dry weight at test end [mg]



Mean



15.7



9.6



12.3



12.4



11.6



10.4



4.3 4)



SD



2.1



1.0



1.02



2.25



3.36



1.21



 -



RSD



13.6



10.3



8.3



18.1



28.9



11.6



 -



3), 4) Williams test, one-sided smaller, p<0.05, compared to the solvent control


Remark: Due to mortality of the juvenile fish, 3 replicates were considered for statistical evaluation of treatment with nominal 158 µg/L, while 1 replicate was considered for the evaluation of nominal 500 µg/L.


 


Table 3:          Summary of NOEC/LOEC and EC50, 20, 10 determination during the course of the study:






















































Endpoint



NOEC / LOEC


nominal concentration


[µg/L]



NOEC / LOEC


Mean measured concentration


[µg/L]



EC10 (95 % CI)


nominal/ mean measured concentration


[µg/L]



EC20 (95 % CI)


nominal/ mean measured concentration


[µg/L]



EC50 (95 % CI)


nominal/ mean measured concentration


[µg/L]



Hatching success



≥500 / >500



≥443 / >443



-1)



-1)



-1)



Post hatch survival,


day 21 pf*



158 / 500



153 / 443



-1)



-1)



-1)



Post hatch survival,


day 35 pf*



50.0 / 158



64.5 / 153



68.9


(26.3-96.0)/


78.4


(11.6-108.4)



96.5


(51.7-122.5)/


103.2


(32.2-130.9)



171.7


(140.5-217.2)/


165.0


(130.9-250.1)



Individual total length*



158 / 500



153 / 443



-1)



-1)



-1)



Individual dry weight*



158 / 500



153 / 443



-1)



-1)



-1)



* Williams Multiple t-test, p<0.05, one-sided smaller


1) not calculated as either no effects were observed or effects were observed only in the highest tested concentration

Validity criteria fulfilled:
yes
Remarks:
Dissolved oxygen between 68 to 102 %; water temperature 26.2 to 26.4 °C; hatching success 100 % in the controls; Post-hatch survival 78.8 to 86.3 % in the controls; Analytic is provided
Conclusions:
The most sensitive endpoint was the fish survival at test end, thus, the NOEC was determined to be 64.5 µg/L and the LOEC to be 153 µg/L (mean measured).
Based on the survival data at test end effect concentration (EC50, 20, 10) calculation was performed by Logit analysis using linear max. likelihood regression. Hereby, EC50, EC20 and EC10 were determined to be 165.0, 103.2 and 78.4 µg/L (mean measured), respectively.
Executive summary:

An early life stage toxicity test with zebrafish (Danio rerio) according to OECD 210 was performed. The study aimed at identifying potential adverse effects of the test item during the early life stage (ELS) of Zebrafish (Danio rerio). The aim of the study was to derive a NOEC (no observed effect concentration). For this reason, early life stages of zebrafish (Danio rerio) were exposed to five concentrations of the test item - 6.05, 21.6, 64.5, 153 and 443 µg/L (mean measured), a solvent control and a water control under flow-through conditions for a period of 35 d. The test was initiated by placing fertilized eggs into test chambers. Hatching rate, mortality, growth, and behavioural abnormalities were recorded.


Since there was a statistically significant difference between control and solvent-control in terms of larval growth, in accordance with OECD 54 (2006), it was decided to choose the solvent-control for statistical evaluations. Statistical evaluation revealed no significant effect of the test item on hatchability, whereas post hatch survival as well as growth in terms of length and weight showed significant statistical differences between solvent-control and treatment with the test item.


The water concentration of the test item was determined using Gas Chromatography (GC) with MS detection. Mean concentrations per treatment of the test item during the course of the study were determined at 6.05, 21.6, 64.5, 153 and 443 µg/L. Thus, these concentrations were between 88.6 % and 136 % of the nominal concentration of the test item. The biological effect concentrations were based on arithmetic mean measured concentrations.


 


Biological effects


Hatching success and post hatch survival


First larvae hatched at day 3 post fertilisation (pf) across all treatment levels. Hatch was completed at day 5 pf in all replicates, with no difference between treatments. Coagulated eggs were not found neither in the controls nor in treatments with the test item.


The mean hatching success was 100 % in control and in the solvent-control. In treatment with 6.05, 21.6, 64.5, 153 and 443 µg/L (mean measured concentration) the mean hatching success was assessed to be between 98.8 and 100 %.


Survival was recorded at day 21 pf and at test end, i.e. at day 35 pf. Mortality of larvae occurred mainly before 21 dpf, during the phase of transition from yolk sac feeding to external feeding.


At test end, the post hatch survival rate in control and solvent-control reached a mean value of 86.3 and 78.8 % and thus met the validity criterion for survival in controls of ≥ 75 %.


The post hatch survival in treatments was determined to be 83.3, 87.5, 90.0, 36.6 and 5.0 % in the test concentrations of 6.05, 21.6, 64.5, 153 and 443 µg/L (mean measured), respectively. There was a statistically significant decrease of survival in treatment with mean measured concentration at 153 µg/L and 443 µg/L (Williams Multiple t-test, one-sided smaller, p<0.05).


 


Individual total length at test termination


The mean total lengths were determined to be 18 mm in the control and 16 mm in the solvent-control. The mean total lengths in the treatment groups were determined to be 18, 17, 17, 17 and 14 mm in test concentrations of 6.05, 21.6, 64.5, 153 and 443 µg/L (mean measured), respectively.


Statistical analyses revealed statistically significant reduction between control and treatment at 443 µg/L (Williams Multiple t-test; p=0.05; one-sided smaller).


 


Individual dry weight at test termination


The mean individual dry weights were determined to be 15.7 mg and 9.6 mg in the control and the solvent-control, respectively. The mean individual dry weights in treatments were assessed to be 12.3, 12.4, 11.6, 10.4 and 4.3 mg at 6.05, 21.6, 64.5, 153 and 443 µg/L (mean measured), respectively.


Statistical analyses revealed a significant reduction compared to solvent control in treatment with 443 µg/L (Williams Multiple t-test; p=0.05; one-sided smaller).


 


Conclusion


Statistical evaluation revealed no significant effect of the test item on hatchability, whereas post hatch survival as well as growth in terms of length and weight showed significant statistical differences between solvent-control and treatment with the test item.


The most sensitive endpoint was the fish survival at test end, thus, the NOEC / LOEC were determined to be 64.5 / 153 µg/L (mean measured).


Based on the survival data at test end effect concentration (EC50, 20, 10) calculation was performed by Logit analysis using linear max. likelihood regression. Hereby, EC50, EC20 and EC10 were determined to be 165.0, 103.2 and 78.4 µg/L (mean measured), respectively.

Description of key information

A fish early life toxicity study with Danio rerio was performed. The most sensitive endpoint was the fish survival at test end, thus the NOEC / LOEC were determined to be 64.5 / 153 µg/L (mean measured).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
NOEC
Effect concentration:
64.5 µg/L

Additional information

An early life stage toxicity test with zebrafish (Danio rerio) according to OECD 210 was performed. The study aimed at identifying potential adverse effects of the test item during the early life stage (ELS) of Zebrafish (Danio rerio). The aim of the study was to derive a NOEC (no observed effect concentration). For this reason, early life stages of zebrafish (Danio rerio) were exposed to five concentrations of the test item - 6.05, 21.6, 64.5, 153 and 443 µg/L (mean measured), a solvent control and a water control under flow-through conditions for a period of 35 d. The test was initiated by placing fertilized eggs into test chambers. Hatching rate, mortality, growth, and behavioural abnormalities were recorded.


Since there was a statistically significant difference between control and solvent-control in terms of larval growth, in accordance with OECD 54 (2006), it was decided to choose the solvent-control for statistical evaluations. Statistical evaluation revealed no significant effect of the test item on hatchability, whereas post hatch survival as well as growth in terms of length and weight showed significant statistical differences between solvent-control and treatment with the test item.


The water concentration of the test item was determined using Gas Chromatography (GC) with MS detection. Mean concentrations per treatment of the test item during the course of the study were determined at 6.05, 21.6, 64.5, 153 and 443 µg/L. Thus, these concentrations were between 88.6 % and 136 % of the nominal concentration of the test item. The biological effect concentrations were based on arithmetic mean measured concentrations.


 


Biological effects


Hatching success and post hatch survival


First larvae hatched at day 3 post fertilisation (pf) across all treatment levels. Hatch was completed at day 5 pf in all replicates, with no difference between treatments. Coagulated eggs were not found neither in the controls nor in treatments with the test item.


The mean hatching success was 100 % in control and in the solvent-control. In treatment with 6.05, 21.6, 64.5, 153 and 443 µg/L (mean measured concentration) the mean hatching success was assessed to be between 98.8 and 100 %.


Survival was recorded at day 21 pf and at test end, i.e. at day 35 pf. Mortality of larvae occurred mainly before 21 dpf, during the phase of transition from yolk sac feeding to external feeding.


At test end, the post hatch survival rate in control and solvent-control reached a mean value of 86.3 and 78.8 % and thus met the validity criterion for survival in controls of ≥ 75 %.


The post hatch survival in treatments was determined to be 83.3, 87.5, 90.0, 36.6 and 5.0 % in the test concentrations of 6.05, 21.6, 64.5, 153 and 443 µg/L (mean measured), respectively. There was a statistically significant decrease of survival in treatment with mean measured concentration at 153 µg/L and 443 µg/L (Williams Multiple t-test, one-sided smaller, p<0.05).


 


Individual total length at test termination


The mean total lengths were determined to be 18 mm in the control and 16 mm in the solvent-control. The mean total lengths in the treatment groups were determined to be 18, 17, 17, 17 and 14 mm in test concentrations of 6.05, 21.6, 64.5, 153 and 443 µg/L (mean measured), respectively.


Statistical analyses revealed statistically significant reduction between control and treatment at 443 µg/L (Williams Multiple t-test; p=0.05; one-sided smaller).


 


Individual dry weight at test termination


The mean individual dry weights were determined to be 15.7 mg and 9.6 mg in the control and the solvent-control, respectively. The mean individual dry weights in treatments were assessed to be 12.3, 12.4, 11.6, 10.4 and 4.3 mg at 6.05, 21.6, 64.5, 153 and 443 µg/L (mean measured), respectively.


Statistical analyses revealed a significant reduction compared to solvent control in treatment with 443 µg/L (Williams Multiple t-test; p=0.05; one-sided smaller).


 


Conclusion


Statistical evaluation revealed no significant effect of the test item on hatchability, whereas post hatch survival as well as growth in terms of length and weight showed significant statistical differences between solvent-control and treatment with the test item.


The most sensitive endpoint was the fish survival at test end, thus, the NOEC / LOEC were determined to be 64.5 / 153 µg/L (mean measured).


Based on the survival data at test end effect concentration (EC50, 20, 10) calculation was performed by Logit analysis using linear max. likelihood regression. Hereby, EC50, EC20 and EC10 were determined to be 165.0, 103.2 and 78.4 µg/L (mean measured), respectively.