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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:
2021-01-12 to 2021-02-15
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
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples of test media including control group were taken from alternating test replicates and the mixing chamber supply of these replicates on days -1, 0 and weekly thereafter until end of exposure. At the end of the exposure the aqueous phase of replicates used for analysis of glassware adsorption were analysed too. The changing intervals of the stock dispersions were taken into account. The stock dispersions were sampled and analyzed from freshly prepared and corresponding 4 days aged stock dispersions of one application interval.

The sorption of the test item on glass was quantified at the loading rates of 4.00 – 16.0 – 64.0 mg/L from one replicate per test group at the end of the exposure (4.00 and 16.0 mg/L) and when 100 % mortality occurred (64.0 mg/L). After sampling of the aqueous phase, the glassware was emptied and an appropriate amount of a suitable solvent was added. The concentration of the test item in this solution was measured and the adsorbed test item amount was calculated from this vessel. Before this procedure, the test vessels were carefully rinsed with dilution water water to remove the remaining dissolved/dispersed test media.

At each sampling day 2 samples were taken per (alternating) test replicate. For each sample 40 mL of each test item loading rate and the control were sampled. For analysis of fresh and aged stock dispersions, approx. 5 mL of each stock dispersion were sampled (two replicates).

Vehicle:
no
Details on test solutions:
Preparation of the Test Item Dispersion
A test item dispersion was made from the original test item. The test item was melted at 90 °C. 40 g of the test item were weighed out and transferred into a glass vessel with 360 mL ultra-pure water. The dispersion was mixed for 15 minutes with an ultraturrax (T25 digital) at 80 – 90 °C at 9500 rpm after addition of the test item.

Dry content 10.3%
Appearance Cream-colored, liquid
Water Solubility Dispersible
pH value Approx. 3 - 4

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebra fish
- Source: All fish used in the test were reared at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin, Germany)

Maintenance of brood fish
A breeding stock of unexposed, mature zebrafish with an age of approx. 10 - 12 months were used for the 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.
- Temperature: 25 ± 2 °C
- Dissolved oxygen concentration > 60% of air saturation value
- pH value: 6 – 8.5
- Photoperiod: 16 h light / 8 h dark cycle
(2 transition periods, 30 minutes each)
- Diffuse light (7 – 750 lux on water surface)
- Food: Artemia salina nauplii, 48 hours old, ad libitum; Daphnia magna, juvenile and adult daphnids, ad libitum; dry food sera vipan SERA, ad libitum.
- No disease treatments were administered. 

Water 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.
Nominal water parameters:
Total hardness: 10 – 250 mg CaCO3/L
pH-value: 6.0 – 8.5
TOC: < 2 mgC/L
Alkalinity: 0.6 mmol/L (recent measurement: 2021-01-14)
Acidity: 0.2 mmol/L (recent measurement: 2021-01-14)
Conductivity: 176 µS/cm (recent measurement: 2021-01-14)

Spawning
15 – 35 adult zebrafish were kept in 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 start of artificial dawning rectangular dishes covered with a stainless-steel mesh and provided with artificial plants (plastic), were introduced into the aquaria. After 1.5 hours the glass dishes were gently removed. Eggs were checked carefully for abnormalities like fungus infections. These eggs as well as coagulated and not fertilized eggs were discarded (less than 30%). About 800 eggs were taken and washed in dilution water. Eggs originated from 3 different spawnings.

Start of exposure
The eggs that were used to start the exposure were pooled and attributed randomly (eggs were placed in alternating groups into each of the test groups) to the test groups in crystallization dishes containing test solutions (two dishes per test group, each dish loaded with at least 60 eggs, resulting in a total of minimum 120 eggs per test item loading rate).

Fertilization check
Immediately after exposing the eggs to the test solutions (start of exposure), the eggs were checked for fertilization. Eggs were fully covered with the respective test solutions. Every embryo was checked under a stereo microscope for its stage. Cleavages which form 4, 8, 16 and 32 cell blastomers could be clearly identified by the development of the blastula and were regarded to be fertilized. Eggs with only a 2 cell stage were regarded as not fertilized and discarded.

Fertilization rate
The mean fertilization rate was 95%.

Introduction of eggs
The eggs were placed onto gauze cages positioned in the middle of the water phase of the test vessels directly after the fertilization check at a stage before cleavage of the blastodisc commences or as close as possible to this stage. The eggs were transferred randomly into test vessels containing the respective exposure solutions. The distribution of eggs to the test item loading rates was carried out indiscriminately by adding 5 eggs to the first test group, the 2nd 5 eggs to the next test group and so on, until all test groups contained the necessary number of eggs.

Feeding of test fish
The feeding regime was ad libitum during the whole feeding period (study day 5 to 34).
Feeding started 2 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 a 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 – 7 times daily). 1 day after start of feeding brine shrimp nauplii (48 h old) were fed until the end of the test (2 – 7 times daily).
Brine shrimp nauplii origin, breeding conditions:
Artemia salina (Brine shrimp eggs) were purchased from Kessler Zoologiegroßhandel GmbH & Co. KG, D 67122 Altrip, Germany. Fresh cultures were prepared with salt water (NaCl 20 g/L, ca. 2 g eggs to 1 L salt water, gentle aeration for 24 - 48 hours at approx. 22 °C). 24 - 48 h old brine shrimp nauplii were harvested, washed in a stainless-steel mesh and resuspended in tap water. Feeding ad libitum was carried out
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
34 d
Remarks on exposure duration:
30 days post hatch
Test temperature:
25.7°C (25.1 - 26.1°C)
pH:
7.44 to 8.05
Dissolved oxygen:
93 – 97% Air Saturation Value (mean)
Nominal and measured concentrations:
4.00 - 8.00 - 16.0 - 32.0 - 64.0 mg test item dispersion/L (separation factor: 2), corresponding to 0.412 - 0.824 - 1.65 - 3.30 - 6.59 mg test item/L (dry content 10.3%)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Size of vessel:
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume:
- Aeration: The dilution water supply tank was aerated. No additional aeration of the test vessels was provided.
- Type of flow-through (e.g. peristaltic or proportional diluter): 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 dispersions. The stock dispersion 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 loading rate and control) where the eggs/fish were exposed.
- Renewal rate of test solution (frequency/flow rate): Water exchange in the test aquaria approx. 5 times per day (1.563 L/h)
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
A fish 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 solutions flowed through the test vessels for 6 days prior to the start of the exposure. The measured concentrations were in the acceptable range of 70 to 109% of the nominal loading rates concentrations, with no trend of increasing or decreasing.

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16 / 8 h photoperiod (light / dark)
- Light intensity: 300 ± 150 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

RANGE-FINDING STUDY
The test loading rates were based on the results of a preliminary range finding test (non-GLP) conducted under flow-through conditions over 15 days.
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
not required
Duration:
30 d
Dose descriptor:
EC10
Effect conc.:
0.582 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Weight
Remarks on result:
other: 95% c.i. 0.287 – 1.18 mg/L
Duration:
30 d
Dose descriptor:
EC10
Effect conc.:
0.66 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
length
Remarks on result:
other: 95% c.i. 0.411 – 1.06 mg/L
Duration:
30 d
Dose descriptor:
EC10
Effect conc.:
1.79 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Hatching success after 6 days
Remarks on result:
other: 95% c.i. 0.581 - > 4.68 mg/L
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
0.224 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: post hatch survival and overall survival
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
0.461 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: fry growth (expressed as length and fresh weight)
Duration:
6 d
Dose descriptor:
NOEC
Effect conc.:
0.461 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: hatching success after 6 d
Details on results:
The mean egg fertilization rate determined on study day 0 (start of the exposure) was 95%.
Hatch began on study day 3 in the control group and all test groups. The hatch of larvae of all test groups was completed until study day 6. Study day 4 was determined to be post hatch day 0 (PHD 0) with a hatching rate of 95% in the control.
The swim-up period of the control and the test item loading rates 4.00 to 16.0 mg dispersion/L was observed from study day 5 to 7. First swim-up of few larvae of the test item loading rates 32.0 and 64.0 mg dispersion/L was observed on study day 5. However completion of swim-up was not observed for these loading rates, since behavioural and non-lethal effects interfered with the swim-up.
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 96% in the control, thus fully meeting the validity criteria of the guideline given in part 10.
A loading-related decrease of the post-hatch survival was detected with increasing test item loading rates.
The cumulative mortality at the end of the exposure, related to the number of eggs introduced on day 0, was 7% in the control group. A loading-related decrease of the overall survival (increase of overall mortality) was detected in the test item loading rates.
No morphological and behavioral effects were observed in the control group. In the two lowest test item loading rates of 4.00 and 8.00 mg dispersion/L non-lethal effects were observed for a short period of 2 to 4 days. The test item loading rate of 16.0 mg dispersion/L showed an increase of observed non-lethal effects with ongoing exposure from study day 10 up to study day 22. The two highest test item loading rates of 32.0 and 64.0 mg dispersion/L showed non-lethal effects after hatch from study day 6 to 12 resulting in 100 % mortality.
Reported statistics and error estimates:
NOEL/LOEL: The data of the parameters hatch, post hatch survival and overall survival were arcsine transformed prior to statistical analysis. Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01. Levene’s test on variance was done with a significance level of 0.01. Monotonicity was done by trend analysis by contrasts (significance level 0.05). The Williams multiple sequential t-test procedure was done with a significance level of 0.05.
All calculations were done with ToxRat Professional and based on the nominal loading rates and the time-weighted arithmetic mean measured concentrations of the test item.

ELx/LLx-calculation: The ELx/LLX-values and the corresponding confidence intervals were calculated by standard procedures with GraphPad Prism (GRAPHPAD SOFTWARE, INC.) and ToxRat Professional (TOXRAT SOLUTIONS GMBH).

Table 1: NOEL, LOEL, ELx Values of Hatching Success and Fry Growth


Based on nominal test item loading rates [mg test item/L] with 95% Confidence intervals in brackets


 













































Parameter



Hatching success after
6 days1)



Fry Growth
expressed as:



Length



Weight



NOEL



0.824



0.824



0.824



LOEL



1.65



1.65



1.65



EL10



2.66
(0.989 - > 6.59)



1.15
(0.745 – 1.77)



1.02
(0.535 – 1.94)



EL20



> 6.59



1.63
(1.02 – 2.61)



1.29
(0.686 – 2.45)



EL50



> 6.59



3.19
(1.24 – > 6.59)



2.02
(0.798 – 4.98)



 



  • end of hatching period


 


Table 2: NOEC, LOEC, ECx Values of Hatching Success and Fry Growth


Based on time weighted arithmetic mean measured concentrations [mg test item/L] with 95% Confidence intervals in brackets


 













































Parameter



Hatching success after
6 days1)



Fry Growth
expressed as:



Length



Weight



NOEC



0.461



0.461



0.461



LOEC



0.982



0.982



0.982



EC10



1.79
(0.581 - > 4.68)



0.660
(0.411 – 1.06)



0.582
(0.287 – 1.18)



EC20



> 4.68



0.968
(0.584 – 1.62)



0.750
(0.376 – 1.52)



EC50



> 4.68



2.01
(0.717 - > 4.68)



1.22
(0.436 – 3.29)




  • end of hatching period


 


 


Table 3: NOEL, LOEL, LLx values of Post Hatch Survival and Overall Survival


Based on nominal test item loading rates [mg test item/L] with 95% Confidence intervals in brackets


 













































Parameter



Post-hatch survival


 



Overall survival


 
 

NOEL



0.412



0.412


 

LOEL



0.824



0.824


 

LL10



n.a.



n.a.


 

LL20



0.784
(< 0.412 – 1.07)



0.707
(< 0.412 – 1.04)


 

LL50



1.21
(0.996 – 1.40)



1.17
(0.949 – 1.38)


 

n.a. = Not applicable


 


 


Table 4: NOEC, LOEC, LCx values of Post Hatch Survival and Overall Survival


Based on time weighted arithmetic mean measured concentrations [mg test item/L] with 95% Confidence intervals in brackets


 













































Parameter



Post-hatch survival


 



Overall survival


 
 

NOEC



0.224



0.224


 

LOEC



0.461



0.461


 

LC10



n.a.



n.a.


 

LC20



0.436
(< 0.224 – 0.609)



0.391
(< 0.224 – 0.594)


 

LC50



0.699
(0.565 – 0.820)



0.674
(0.536 – 0.808)


 

n.a. = Not applicable


 


 


The study was performed according to OECD 210 and met the validity criteria:


 



  • Dissolved oxygen saturation was between 68 and 100% of the air saturation value
    (required: > 60%).

  • Water temperature was in the recommended range for the test species (i.e. 26 °C ±5 °C): 25.1 – 26.1 °C. It did not differ by more than ± 1.5 °C between test vessels or between successive days at any time during the test.

  • Hatching success was 95% in the control group (required: ≥ 70%).

  • Post-hatch survival was 96% in the control group (required: ≥ 75%).


Concentration analysis: the concentrations of the test item in the test solutions should be maintained within 80 – 120% of the nominal values. Since the test item is an UVCB substance all effect values were given based on the nominal loading rates. Additionally, all endpoints were presented based on the time weighted arithmetic mean measured concentrations of the test item.

Validity criteria fulfilled:
yes
Conclusions:
Partially unsaturated TEA-Esterquat caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal loading rates of 0.412, 0.824, 1.65, 3.30 and 6.59 mg test item/L, corresponding to the time weighted arithmetic mean measured (TWA) concentrations of 0.224, 0.461, 0.982, 2.60 and 4.68 mg test item/L.
For the parameter hatch, the NOEL was 0.824 mg test item/L nominal (TWA: 0.461 mg test item/L). Therefore, the respective LOEL was determined to be 1.65 mg test item/L nominal (TWA: 0.982 mg test item/L).
For the parameters post hatch survival and overall survival, the NOELs were 0.412 mg test item/L nominal (TWA: 0.224 mg test item/L), respectively. Therefore, the respective LOELs were determined to be 0.824 mg test item/L nominal (TWA: 0.461 mg test item/L).
For the parameter fry growth (expressed as length and fresh weight) the NOELs were 0.824 mg test item/L nominal (TWA: 0.461 mg test item/L) for both parameters. Therefore, the LOELs for length and weight were determined to be 1.65 mg test item/L nominal (TWA: 0.982 mg test item/L), respectively.
Executive summary:

The effects of the test item partially unsatd TEA-Esterquat on the early-life stage of fish (Danio rerio/ Zebrafish) were determined according to OECD Guideline 210.


 


A test item dispersion with a dry content of 10.3% was prepared from the original test item. Stock dispersions in demineralized water with nominal concentrations of 8.00, 16.0, 32.0, 64.0 and 128 g dispersion/L, corresponding to 0.824, 1.65, 3.30, 6.59 and 13.2 g test item/L were prepared in appropriate intervals of 3 to 4 days and continuously dosed to the dilution water in a flow-through system. Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item loading rates of 4.00, 8.00, 16.0, 32.0 and 64.0 mg dispersion/L, corresponding to 0.412, 0.824, 1.65, 3.30 and 6.59 mg test item/L ), corresponding to time weighted arithmetic mean measured concentrations of 0.224, 0.461, 0.982, 2.60 and 4.68 mg test item/L..


The test was started by placing fertilized eggs into the test vessels and it lasted 34 days (30 days post-hatch). 80 eggs of Danio rerio/ zebrafish were exposed to each test item loading rate and the control (4 replicates with 20 eggs each).


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


On study day 4, 95% of the control larvae had hatched. Therefore, study day 4 was defined as post hatch day 0 (= PHD 0).


Different 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.


Specific analysis of various concentrations ofthe test item in the test media and the controls was carried out via LC-MS/MS.


The test media were sampled and analyzed from alternating test vessels prior to exposure on day -1 and during the exposure on study days 0, 7, 14, 21 and 28.


The measured concentrations of the test media during equilibration days -3 to -1 ranged between 70 and 109% of the nominal loading rates. The measured concentrations of the test media on study days 0 to 28 were in the range of 31 to 88% of the nominal loading rates.


The stock dispersions were sampled and analyzed from freshly prepared and corresponding 4 days aged stock dispersions. Measured concentrations of the freshly prepared stock dispersions were 92 to 99% of the nominal values. Measured concentrations of the 4 days aged stock dispersions in the range of 91 to 99% of the nominal values.


 


At the end of the exposure on study day 34 the aqueous phase of replicates used for analysis of glassware adsorption were analysed too. The sorption of the test item on glass was quantified at the loading rates of 4.00 and 16.0 mg dispersion/L from one replicate per test group at the end of the exposure and from one replicate the loading rate of 64 mg dispersion/L after 100 % mortality occurred.


 


Findings and Observations


The results of the parameters hatching success, fry growth (expressed as weight and length measurement at PHD 30), post-hatch survival and overall survival were checked for statistically significant differences.


The test item caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal loading rates of 0.412, 0.824, 1.65, 3.30 and 6.59 mg test item/L, corresponding to the time weighted arithmetic mean measured (TWA) concentrations of 0.224, 0.461, 0.982, 2.60 and 4.68 mg test item/L.


For the parameter hatch, the NOEL was 0.824 mg test item/L (TWA: 0.461 mg test item/L). Therefore, the respective LOEL was determined to be 1.65 mg test item/L (TWA: 0.982 mg test item/L).


For the parameters post hatch survival and overall survival, the NOELs were 0.412 mg test item/L (TWA: 0.224 mg test item/L), respectively. Therefore, the respective LOELs were determined to be 0.824 mg test item/L (TWA: 0.461 mg test item/L).


For the parameter fry growth (expressed as length and fresh weight) the NOELs were 0.824 mg test item/L (TWA: 0.461 mg test item/L) for both parameters. Therefore, the LOELs for length and weight were determined to be 1.65 mg test item/L (TWA: 0.982 mg test item/L), respectively.

Description of key information

NOEC (Post-hatch survival, overall survival) = 0.224 (OECD TG 210, Danio rerio); GLP; RL1 (weighted arithmetic mean measured concentrations)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
NOEC
Effect concentration:
0.224 mg/L

Additional information

The effects of the test item partially unsatd TEA-Esterquat on the early-life stage of fish (Danio rerio/ Zebrafish) were determined according to OECD Guideline 210.


 


A test item dispersion with a dry content of 10.3% was prepared from the original test item. Stock dispersions in demineralized water with nominal concentrations of 8.00, 16.0, 32.0, 64.0 and 128 g dispersion/L, corresponding to 0.824, 1.65, 3.30, 6.59 and 13.2 g test item/L were prepared in appropriate intervals of 3 to 4 days and continuously dosed to the dilution water in a flow-through system. Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item loading rates of 4.00, 8.00, 16.0, 32.0 and 64.0 mg dispersion/L, corresponding to 0.412, 0.824, 1.65, 3.30 and 6.59 mg test item/L ), corresponding to time weighted arithmetic mean measured concentrations of 0.224, 0.461, 0.982, 2.60 and 4.68 mg test item/L..


The test was started by placing fertilized eggs into the test vessels and it lasted 34 days (30 days post-hatch). 80 eggs of Danio rerio/ zebrafish were exposed to each test item loading rate and the control (4 replicates with 20 eggs each).


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


On study day 4, 95% of the control larvae had hatched. Therefore, study day 4 was defined as post hatch day 0 (= PHD 0).


Different 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.


Specific analysis of various concentrations ofthe test item in the test media and the controls was carried out via LC-MS/MS.


The test media were sampled and analyzed from alternating test vessels prior to exposure on day -1 and during the exposure on study days 0, 7, 14, 21 and 28.


The measured concentrations of the test media during equilibration days -3 to -1 ranged between 70 and 109% of the nominal loading rates. The measured concentrations of the test media on study days 0 to 28 were in the range of 31 to 88% of the nominal loading rates.


The stock dispersions were sampled and analyzed from freshly prepared and corresponding 4 days aged stock dispersions. Measured concentrations of the freshly prepared stock dispersions were 92 to 99% of the nominal values. Measured concentrations of the 4 days aged stock dispersions in the range of 91 to 99% of the nominal values.


 


At the end of the exposure on study day 34 the aqueous phase of replicates used for analysis of glassware adsorption were analysed too. The sorption of the test item on glass was quantified at the loading rates of 4.00 and 16.0 mg dispersion/L from one replicate per test group at the end of the exposure and from one replicate the loading rate of 64 mg dispersion/L after 100 % mortality occurred.


 


Findings and Observations


The results of the parameters hatching success, fry growth (expressed as weight and length measurement at PHD 30), post-hatch survival and overall survival were checked for statistically significant differences.


The test item caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal loading rates of 0.412, 0.824, 1.65, 3.30 and 6.59 mg test item/L, corresponding to the time weighted arithmetic mean measured (TWA) concentrations of 0.224, 0.461, 0.982, 2.60 and 4.68 mg test item/L.


For the parameter hatch, the NOEL was 0.824 mg test item/L (TWA: 0.461 mg test item/L). Therefore, the respective LOEL was determined to be 1.65 mg test item/L (TWA: 0.982 mg test item/L).


For the parameters post hatch survival and overall survival, the NOELs were 0.412 mg test item/L (TWA: 0.224 mg test item/L), respectively. Therefore, the respective LOELs were determined to be 0.824 mg test item/L (TWA: 0.461 mg test item/L).


For the parameter fry growth (expressed as length and fresh weight) the NOELs were 0.824 mg test item/L (TWA: 0.461 mg test item/L) for both parameters. Therefore, the LOELs for length and weight were determined to be 1.65 mg test item/L (TWA: 0.982 mg test item/L), respectively.