Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Ecotoxicological information

Long-term toxicity to fish

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 March 2021 - 04 April 2021
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
Qualifier:
according to guideline
Guideline:
other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD Series on Testing and Assessment no. 23
Version / remarks:
2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 6.0, 9.5, 15.3, 24.4, 39.1, 62.5 and 100.0 mg/L.
- Sampling method: During the main study, samples from all the test concentrations were collected. Fresh media samples were collected at 0 hours on days 6, 8, 14, 16 and 18; spent media were be collected on days 8, 10, 16, 18 and 20, and they were analysed for active content. Before collection, the test media was mixed slowly (without affecting the test system) and then, sampling was made; 10 mL water samples were collected from the central point of the tank in duplicates from all the test concentrations. Additionally, at 0, 24, and 48 hours, three samples (about 15 ml each) were collected in duplicates from test media replicates for analytical verification of saturation concentration.
- Sample storage conditions before analysis: One set was analysed on the same day of collection and the other set was stored at 26 ± 1.5ºC as backup sample.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item stock solution was prepared by weighing required quantity of the test item in beaker. To this small volume of reverse osmosis water was added and stirred well, after complete solubility, the test formulation was transferred to measuring cylinder and beaker was rinsed with reverse osmosis water and transferred again to measuring cylinder, the rinsing procedure was repeated until the complete transfer of test contents, finally the volume made up to required volume using reverse osmosis water. The solution prepared was kept on magnetic stirrer to maintain homogeneity. The stock solution prepared was introduced to know volume of dilution medium to obtain desired concentration. The preparation of test media on day 0 during main study are presented in ''Any other information on materials and methods incl. tables.
- Test concentration separation factor: 1.6
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test media was clear with no fine particles on the day of preparation (fresh) and the appearance of test media was pink in all the test concentration. However, at 24 and 48 hours (spent) observation fine particles were settled at the bottom of the test vessels at the test concentrations of 92.5 and 100.0 mg/L.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Strain: Danio rerio
- Source: Fertilized eggs of Danio rerio were procured from Whizbang Bioresearch Private Limited, Tamil Nadu, India.

POST-HATCH FEEDING
- Type/source of feed: flake food (commercial larvae food or BSN48 flake food for juvenile fish as appropriate)
- Frequency of feeding: twice daily
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 d
Hardness:
168 to 216 mg/L as CaCO3
pH:
6.01 to 6.98
Dissolved oxygen:
80.9 to 98.8%
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 6.0, 9.5, 15.3, 24.4, 39.1, 62.5 and 100.0 mg/L.
Measured concentrations: see table in 'any other information on results incl. tables'. The test concentrations were maintained at ± 20% recovery to nominal concentration throughout the exposure period.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass aquarium vessels
- Material, size, headspace, fill volume: glass, 3-7L fill volume
- Renewal rate of test solution (frequency/flow rate): 48 hours
- No. of fertilized eggs/embryos per vessel: 20 eggs per vessel, 80 eggs per group
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: Maximum loading of fish embryos did not exceed 0.5 g/L wet weight per 24 hours solution, and not exceeding 5 g/L of solution at any time, as recommended.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis water (from tap water)
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen and pH were measured in all the test vessels at the beginning and at every renewal period, hardness at the beginning and end of the test. Temperature was monitored daily until the termination of the test from all the test vessels.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 - 16 hours (constant light regime)
- Light intensity: n.a.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The embryonic stage at the beginning of exposure to the test item was verified using a representative sample of eggs suitably preserved and cleared. Observation for Hatching and survival was made daily, and numbers were recorded. Dead embryos, larvae and juvenile fish were removed as soon as observed. If fungus were detected on eggs in early embryonic development, those eggs were counted and removed.

RANGE-FINDING STUDY (n.a.)
- Results used to determine the conditions for the definitive study: The test concentrations for the main study have been selected based on the available results from read across data from The test concentrations for the main study were selected based on the available results from Acute Toxicity Test of C.I. Pigment Blue 29 to Oryzias latipes by MITI (Blue Sodium polysulfide aluminosilicate with a SOD-type framework structure), 96 hours LC50 is >90 mg/L.
Reference substance (positive control):
not required
Duration:
30 d
Dose descriptor:
EC10
Effect conc.:
17.41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Remarks on result:
other: 95% CL 14.81 - 20.48
Duration:
30 d
Dose descriptor:
EC10
Effect conc.:
14.52 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 8.27 - 25.5
Duration:
30 d
Dose descriptor:
EC10
Effect conc.:
8.72 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Remarks on result:
other: 95% CL: 7.01 - 10.84
Key result
Duration:
30 d
Dose descriptor:
EC10
Effect conc.:
7.46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Remarks on result:
other: 95% CL: 4.11 - 13.56
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
15.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
15.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
9.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: Post hatch larval survival until day 30 in the control group was 95.8%. Post-hatch larval survival across the test concentrations ranges from 91.5, 90.0, 87.9, 83.9, 80.8, 48.8 and 11.8% at the tested concentrations of 6.0, 9.5, 15.3, 24.4, 39.1, 62.5 and 100.0 mg/L, respectively.
- Days to hatch or time to release of young: First egg hatch in control and treatment vessels occurred at 48 hour period.
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: Hatching success of 88.8, 87.5, 82.5, 77.5, 65.0, 51.3 and 21.3% was observed at the tested concentrations of 6.0, 9.5, 15.3, 24.4, 39.1, 62.5 and 100.0 mg/L, respectively.
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): n.a.
- Observations on body length and weight of young and/or exposed parents at one or more time periods: Total length was measured for the surviving fish at the termination of the test, mean fish length measured for found to be 2.9 cm in control, 2.8, 2.5, 2.0, 1.8, 1.4, 1.1 and 0.5 cm in the tested concentrations of 6.0, 9.5, 15.3, 24.4, 39.1, 62.5 and 100.0 mg/L, respectively.
- Number of healthy fish at end of test: n.a.
- Type of and number with morphological abnormalities: No abnormal appearance was observed in the control and the tested concentration of 6.0 mg/L. Abnormal appearance of bent tail (tail malformations) was observed at the tested concentrations of 9.5, 15.3, 39.1 and 62.5 mg/L. Abnormal appearance of bent tail (tail malformation) and darkened coloration was observed at the test concentration of 100.0 mg/L. Tail malformation (bent tail) was considered as sub-lethal effects of test item.
- Type of and number with behavioural abnormalities: No abnormal behaviour was observed in the control or the 6.0 mg/L group. Abnormal behaviour of fish localized on the bottom of the test vessel with no active movement was observed at the concentration of 9.5 mg/L and 100.0 mg/L.
Erratic behaviour and fish localized on the bottom of the test vessel with no active movement was observed at the tested concentrations of 15.3, 24.4, 39.1, and 62.5 mg/L.
- Type and number of developmental effects: Embryonic stage was verified from the representative samples using a microscope, stage was in the before cleavage of the blastodiscs commences eggs were clear with any abnormalities.
- Effect concentrations exceeding solubility of substance in test medium: no. The limit of saturation of test item in test media was determined to be 11.04 to 13.05 mg/L.
- Incidents in the course of the test which might have influenced the results: none reported.
Reported statistics and error estimates:
Statistical analysis was performed to determine the NOEC and LOEC for cumulative mortality, numbers of healthy fish at end of test, length and weight of surviving fishes, numbers of deformed larvae, number of survival larvae and numbers of fish exhibiting abnormal behaviour. In order to make a multiple comparison between the results at the individual concentrations and those for the controls Dunnett’s method was employed.
EC10, EC20 and EC50 was calculated using Probit Finney’s method for larvae hatching success, larvae survival, fish length and fish wet weight.

Table 1. Summary of results.









































































































































Parameter



Endpoint (mg/L)



EC10



17.41



EC20



27.15



EC50



63.50



Larvae Hatching


Success



Lower limit



14.81



Lower limit



24.01



Lower limit



55.67



Upper Limit



20.48



Upper Limit



30.71



Upper Limit



72.45



 



 



 



 



 



 



 



Larvae Surviving



EC10



14.52



EC20



23.51



EC50



59.14



Lower limit



8.27



Lower limit



15.58



Lower limit



39.93



Upper Limit



25.5



Upper Limit



35.48



Upper Limit



87.58



 



 



 



 



 



 



 



Fish Length



EC10



8.72



EC20



14.2



EC50



36.11



Lower limit



7.01



Lower limit



12.05



Lower limit



31.75



Upper Limit



10.84



Upper Limit



16.73



Upper Limit



41.08



 



 



 



 



 



 



 



Fish fresh weight



EC10



7.46



EC20



11.55



EC50



26.65



Lower limit



4.11



Lower limit



7.28



Lower limit



19.09



Upper Limit



13.56



Upper Limit



18.33



Upper Limit



37.2



 


































Parameters



End Points (mg/L)



NOEC



LOEC



Larvae Hatching Success



15.3



24.4



Larvae Surviving



15.3



24.4



Fish Length



9.5



15.3



Fish Fresh Weight



6.0



9.5



 


Table 2. Post-hatch observed larval fish mortality

































































































































































































































































































































































































































































































































































































































































































 



Group



Test Conc. (mg/L)



R



 



Days



 



2



4



6



8



10



12



14



16



18



20



22



24



26



28



30



32



 



G1



0.0



1



H-18



N-18



N-18



N-18



N-18



N-17, D-1



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



 



2



H-19



N-19



N-19



N-19



N-19



N-19



N-19



N-19



N-18, D-1



N-18



N-18



N-18



N-18



N-18



N-18



N-18



 



3



H-18



N-18



N-18



N-18



N-18



N-18



N-18



N-18



N-18



N-18



N-18



N-18



N-18



N-18



N-18



N-18



 



4



H-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-16, D-1



N-16



N-16



N-16



N-16



N-16



N-16



N-16



 



G2



6.0



1



H-18



N-18



N-18



N-18



N-18



N-18



N-17, B-1



N-17, D-1



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



 



2



H-17



N-17



N-17



N-17



N-17



N-17



N-17



N-15, D-2



N-15



N-15



N-15



N-15



N-15



N-15



N-15



N-15



 



3



H-19



N-19



N-19



N-19



N-19



N-19



N-19



N-19



N-18, D-1



N-18



N-18



N-18



N-18



N-18



N-18



N-18



 



4



H-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-15, D-2



N-15



N-15



N-15



N-15



N-15



N-15



N-15



 



G3



9.5



1



H-18



N-18



N-18



N-18



N-18



N-18



N-17, D-1



N-16, D-1



N-16, D-1



N-16



N-16



N-14, B-2



N-14, B-2



N-14, B-2



N-14, B-2



N-14, B-2



 



2



H-18



N-18



N-18



N-18



N-18



N-18



N-18



N-18



N-17,     D-1



N-17



N-17



N-17



N-15,  BT-2



N-15,  BT-2



N-15, BT-2



N-15, BT-2



 



3



H-16



N-16



N-16



N-16



N-16



N-16



N-16



N-16



N-16



N-14,D-2



N-14



N-14



N-14



N-14



N-14



N-14



 



4



H-18



N-18



N-18



N-18



N-18



N-18



N-18



N-18



N-18



N-18



N-16,   D-2



N-16



N-15, B-1



N-15, B-1



N-15, B-1



N-15, B-1



 



G4



15.3



1



H-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



N-17



 



2



H-15



N-15



N-15



N-15



N-12, D-3



N-10, B-2



N-10, B-2



N-10, B-2



N-10, B-2



N-10, B-2



N-10, B-2



N-10,  BT-2



N-10,  BT-2



N-10,  BT-2



N-10,  BT-2



N-10,  BT-2



 



3



H-16



N-16



N-16



N-16



N-16



N-12, D-4



N-12



N-10, B-2



N-10, B-2



N-10, B-2



N-10, B-2



N-10, B-2



N-10, B-2



N-9, EB-3



N-9, EB-3



N-9, EB-3



 



4



H-18



N-18



N-18



N-18



N-18



N-18



N-18



N-17, D-1



N-17



N-17



N-15, B-2



N-15, B-2



N-15, EB-2



N-15, EB-2



N-15, EB-2



N-15, EB-2



G5



24.4



1



H-17



N-17



N-17



N-17



N-17



N-17



N-15, B-2



N-12, B-3,     D-2



N-12, B-3



N-12, B-3



N-12, B-3



N-10, B-3, EB-2



N-10, B-3, EB-2



N-10, B-3, EB-2



N-10, B-3, EB-2



N-10, B-3, EB-2



2



H-15



N-15



N-15



N-15



N-15



N-15



N-14, D-1



N-10,      B-4



N-10,   B-4



N-10,    B-4



N-10,  B-4



N-10,  B-4



N-10, B-4



N-10, B-4



N-10,  B-4



N-10,  B-4



3



H-16



N-16



N-16



N-16



N-16



N-16



N-10,   B-6



N-10, B-6



N-8,   B-4,   D-4



N-8,   B-4



N-8,   B-4



N-8,    B-4



N-8,    B-4



N-8,   B-4



N-8,     B-4



N-8,     B-4



4



H-14



N-14



N-14



N-14



N-14



N-14



N-14



N-14



N-14



N-14



N-10, B-1,    D-3



N-10, B-1



N-7,    B-2, EB-2



N-7,   B-2, EB-2



N-7,          B-2, EB-2



N-7,          B-2, EB-2



G6



39.1



1



H-12



N-12



N-12



N-8,    B-2,           D-2



N-8,  B-2



N-8,    B-2



N-8,  B-2



N-8,   B-2



N-6,       B-2, EB-2



N-6,                  B-2, EB-2



N-6,   B-2, EB-2



N-6,   B-2, EB-2



N-6,   B-2, EB-2



N-6,   B-2, EB-2



N-6,   B-2, EB-2



N-6,   B-2, EB-2



2



H-15



N-15



N-15



N-15



N-10, B-2,D-3



N-8,      B-4



N-6,          B-6



N-6,           B-6



N-6,    B-6



N-6,        B-6



N-2,   B-6, EB-4



N-2,           B-6, EB-4



N-2,             B-6, EB-4



N-2,           B-6, EB-4



N-2,           B-6, EB-4



N-2,           B-6, EB-4



3



H-13



N-13



N-13



N-10, B-1,     D-2



N-8,   B-3



N-8,    B-3



N-8,    B-3



N-8,    B-3



N-8,   B-3



N-2,   B-5, EB-4



N-2,   B-5, EB-4



N-2,   B-5, EB-4



N-2,   B-5, EB-4



N-2,   B-5, EB-4



N-2,   B-5, EB-4



N-2,   B-5, EB-4



4



H-12



N-12



N-12



N-12



N-12



N-5,    B-4,   D-3



N-5,    B-4



N-5,   B-4



N-5,   B-4



N-3,   B-4,   BT-2



N-3,   B-4,   BT-2



N-3,   B-4, BT-2



N-3,   B-4,   BT-2



N-3,   B-4,     BT-2



N-3,            B-4, BT-2



N-3,            B-4, BT-2



G7



62.5



1



H-11



N-11



N-10                D-1



N-8                              D-2



N-8



N-2,          B-6



N-2,         B-6



N-2,          B-6



N-2,          B-6



B-4, EB-3, BT-1



B-4, EB-3, BT-1



B-4, EB-3, BT-1



B-4, EB-3, BT-1



B-4, EB-3, BT-1



B-4, EB-3, BT-1



B-4, EB-3, BT-1



2



H-9



N-9



N-4,            B-3,           D-2



B-4,     D-3



B-4



B-2, EB-2



B-2, EB-2



B-2, EB-2



B-2, EB-2



B-2, EB-2



BT-2, EB-2



BT-2, EB-2



BT-2, EB-2



BT-2, EB-2



BT-2, EB-2



BT-2, EB-2



3



H-10



N-10



D-2,            N-8



D-3,          N-5



N-5



B-5



B-5



B-5



B-2, EB-3



B-2, EB-3



B-2, EB-3



BT-2, EB-3



BT-2, EB-3



BT-2, EB-3



BT-2, EB-3



BT-2, EB-3



4



H-11



N-11



N-8,           D-3



B-4,         D-4



B-3,             D-1



B-3



B-3



B-3



B-3



B-1, EB-2



B-1, EB-2



B-1, EB-2



BT-2, EB-1



BT-2, EB-1



BT-2, EB-1



BT-2, EB-1



G8



100.0



1



H-3



B-3



B-3



B-3



B-3



B-3



B-3



B-3



B-3



B-3



B-1,               D-2



DC, B-1



DC, B-1



DC, B-1



DC, B-1



DC, B-1



2



H-5



B-3,            D-2



B-3



B-3



B-3



B-3



B-3



B-3



B-3



B-1,     D-2



B-1



B-1



B-1



B-1



B-1



D-1



3



H-4



B-4



B-4



B-4



B-4



B-4



B-4



B-4



B-1,            D-3



B-1



B-1



DC, B-1



DC, B-1



DC, B-1



DC, B-1



DC, B-1



4



H-5



B-5



B-5



B-5



B-5



B-5



B-2,                  D-3



B-2



B-2



B-2



B-2



B-2



B-2



B-1,               D-1



B-1



D-1



R: Replicate; H: Hatched; N: Normal; B: On bottom; BT: Bent Tail; EB: Erratic behaviour; D: Dead; Alphabet followed by hyphen – indicates no. of larvae exhibiting particular symptom.


 


Table 3. Hatching success, fish larval survival and percent mortality

































































































































































































































































Group



Test Conc. (mg/L)



R



Number of eggs added at the start of the test



Number of hatched larvae



Percentage of hatched larvae



Number of larvae surviving at 30 days post-hatch



Percentage of larvae surviving at 30 days post hatch



Mortality Percent



Individual Vessels



Polled                     vessels



Individual Vessels



Polled vessels



Individual Vessels



Polled vessels



Individual Vessels



Polled                 vessels



Individual Vessels



Polled vessels



No. of Mortality



Polled vessels



% Mortality



G1



0.0



1



20



80



18



72



90



90.0



17



69



94



95.8



1



3



4.2



2



20



19



95



18



95



1



3



20



18



90



18



100



0



4



20



17



85



16



94



1



G2



6.0



1



20



80



18



71



90



88.8



17



65



94



91.5



1



6



8.5



2



20



17



85



15



88



2



3



20



19



95



18



95



1



4



20



17



85



15



88



2



G3



9.5



1



20



80



18



70



90



87.5



16



63



89



90.0



2



7



10.0



2



20



18



90



17



94



1



3



20



16



80



14



88



2



4



20



18



90



16



89



2



G4



15.3



1



20



80



17



66



85



82.5



17



58



100



87.9



0



8



12.1



2



20



15



75



12



80



3



3



20



16



80



12



75



4



4



20



18



90



17



94



1



G5



24.4



1



20



80



17



62



85



77.5



15



52



88



83.9



2



10



16.1



2



20



15



75



14



93



1



3



20



16



80



12



75



4



4



20



14



70



11



79



3














































































































































G6



39.1



1



20



80



12



52



60



65.0



10



42



83



80.8



2



10



19.2



2



20



15



75



12



80



3



3



20



13



65



11



85



2



4



20



12



60



9



75



3



G7



62.5



1



20



80



11



41



55



51.3



8



20



73



48.8



3



21



51.2



2



20



9



45



4



44



5



3



20



10



50



5



50



5



4



20



11



55



3



27



8



G8



100.0



1



20



80



3



17



15



21.3



1



2



33



11.8



2



15



88.2



2



20



5



25



0



0



5



3



20



4



20



1



25



3



4



20



5



25



0



0



5



 


Table 4. Physico-chemical parameters during exposure















































































































































































Group



Test Conc. (mg/L)



Dissolved Oxygen                                               (%)



Temperature                                                     (°C)



pH



Fresh



Spent



Fresh



Spent



Fresh



Spent



Min



Max



Min



Max



Min



Max



Min



Max



Min



Max



Min



Max



G1



0.0



89.90



98.60



81.90



91.90



25.0



25.9



25.1



25.9



6.09



6.78



6.03



6.87



G2



6.0



89.50



98.10



81.70



90.60



25.1



25.9



25.0



25.9



6.01



6.94



6.16



6.88



G3



9.5



88.40



98.80



80.90



90.60



25.0



25.9



25.1



25.9



6.05



6.96



6.34



6.91



G4



15.3



89.00



96.90



81.40



91.30



25.1



25.9



25.0



25.9



6.03



6.98



6.31



6.88



G5



24.4



86.20



96.30



81.40



92.60



25.0



25.9



25.1



25.9



6.27



6.86



6.29



6.85



G6



39.1



89.70



96.70



81.80



92.30



25.1



25.9



25.1



25.9



6.22



6.90



6.25



6.90



G7



62.5



89.30



97.90



82.70



89.60



25.1



25.9



25.1



25.9



6.29



6.96



6.09



6.84



G8



100.0



89.90



96.40



82.10



90.70



25.1



25.9



25.1



25.9



6.24



6.94



6.32



6.86



Min



80.90



25.0



6.01



Max



98.80



25.9



6.98



 


Table 5. Percent inhibition in larvae hatching, survival, length and fresh weight














































































Group



Test Conc. (mg/L)



Percent Inhibition in Larvae Hatching



Percent Inhibition in Post hatch Larvae Surviving



Percent Inhibition of Larvae Length



Percent Inhibition of Fish Fresh Weight



G1



0.0



-



-



-



-



G2



6.0



1.39



4.47



3.5



8.05



G3



9.5



2.78



6.09



13.1



23.24



G4



15.3



8.33



8.30



28.8



31.05



G5



24.4



13.89



12.48



38.1



35.81



G6



39.1



27.78



15.72



52.0



40.82



G7



62.5



43.06



49.10



60.8



79.86



G8



100.0



76.39



87.72



84.3



96.98



-: Not applicable; Percent inhibition calculated in comparison with the control group


 


Table 6. Summary of test concentrations during main study































































Fresh (Day 6)



Group



Nominal concentration


(mg/L)



 Conc. obtained                        


 (mg/L)



Recovery


(%)



G1



0.0



-



-



G2



6.0



5.655



94.26



G3



9.5



9.059



95.36



G4



15.3



14.311



93.67



G5



24.4



23.831



97.67



G6



39.1



37.075



94.82



G7



62.5



62.934



100.69



G8



100.0



97.732



97.73



 































































Spent (Day 8)



Group



Nominal concentration


(mg/L)



 Conc. obtained                        


 (mg/L)



Recovery


(%)



G1



0.0



-



-



G2



6.0



5.605



93.41



G3



9.5



8.878



93.45



G4



15.3



15.409



100.71



G5



24.4



25.910



106.19



G6



39.1



39.255



100.40



G7



62.5



67.212



107.54



G8



100.0



103.475



103.47



 































































Fresh (Day 8)



Group



Nominal concentration


(mg/L)



 Conc. obtained                        


 (mg/L)



Recovery


(%)



G1



0.0



-



-



G2



6.0



5.821



97.02



G3



9.5



9.105



95.85



G4



15.3



14.289



93.39



G5



24.4



24.455



100.23



G6



39.1



36.666



93.77



G7



62.5



63.676



101.88



G8



100.0



100.072



100.07
































































Spent (Day 10)



Group



Nominal concentration


(mg/L)



 Conc. obtained                        


 (mg/L)



Recovery


(%)



G1



0.0



-



-



G2



6.0



5.499



91.66



G3



9.5



8.929



93.99



G4



15.3



14.247



93.12



G5



24.4



24.384



99.93



G6



39.1



36.829



94.19



G7



62.5



62.842



100.55



G8



100.0



98.094



98.09



 































































Fresh (Day 14)



Group



Nominal concentration


(mg/L)



 Conc. obtained                        


 (mg/L)



Recovery


(%)



G1



0.0



-



-



G2



6.0



5.529



92.15



G3



9.5



8.521



89.69



G4



15.3



14.040



91.76



G5



24.4



23.855



97.77



G6



39.1



36.521



93.40



G7



62.5



62.879



100.60



G8



100.0



94.788



94.79



 































































Spent (Day 16)



Group



Nominal concentration


(mg/L)



 Conc. obtained                        


 (mg/L)



Recovery


(%)



G1



0.0



-



-



G2



6.0



5.61



93.86



G3



9.5



8.677



91.34



G4



15.3



13.935



91.08



G5



24.4



23.475



96.21



G6



39.1



35.521



90.85



G7



62.5



60.973



97.56



G8



100.0



94.136



94.14



 































































Fresh (Day 16)



Group



Nominal concentration


(mg/L)



 Conc. obtained                        


 (mg/L)



Recovery


(%)



G1



0.0



-



-



G2



6.0



5.786



96.43



G3



9.5



8.867



93.34



G4



15.3



14.359



93.85



G5



24.4



23.796



97.52



G6



39.1



36.720



93.91



G7



62.5



62.440



99.90



G8



100.0



95.849



95.85
































































Spent (Day 18)



Group



Nominal concentration


(mg/L)



 Conc. obtained                        


 (mg/L)



Recovery


(%)



G1



0.0



-



-



G2



6.0



5.411



90.18



G3



9.5



8.618



90.71



G4



15.3



13.847



90.50



G5



24.4



22.917



93.92



G6



39.1



35.612



91.08



G7



62.5



60.918



96.99



G8



100.0



92.809



92.81



 































































Fresh (Day 18)



Group



Nominal concentration


(mg/L)



 Conc. obtained                        


 (mg/L)



Recovery


(%)



G1



0.0



-



-



G2



6.0



5.495



91.59



G3



9.5



8.693



91.50



G4



15.3



13.812



90.27



G5



24.4



22.804



93.46



G6



39.1



34.722



88.80



G7



62.5



59.568



95.31



G8



100.0



96.573



96.57



 































































Spent (Day 20)



Group



Nominal concentration


(mg/L)



 Conc. obtained                        


 (mg/L)



Recovery


(%)



G1



0.0



-



-



G2



6.0



5.602



93.37



G3



9.5



8.779



92.41



G4



15.3



14.184



92.71



G5



24.4



23.754



97.35



G6



39.1



36.184



92.54



G7



62.5



61.236



97.98



G8



100.0



94.643



97.64



 


Table 7. Analytical verification of limit of saturation






























































































































Day 0 Analysis


   

Sample Code



Conc. Obtained (mg/L)



Nominal Conc.(mg/L)



% Recovery



Sample G1



-



-



-



Sample G2 As such formulation



88.958



100.000



88.96



Sample G2 G Filtered formulation



12.510



100.000



12.51



Sample G2 Centrifuged formulation



13.050



100.000



13.05


    

Day 1 Analysis


   

Sample Code



Conc. Obtained (mg/L)



Nominal Conc.


(mg/L)



% Recovery



Sample G1



-



-



-



Sample G2 As such formulation



85.174



100.000



85.17



Sample G2 G Filtered formulation



12.529



100.000



12.53



Sample G2 Centrifuged formulation



11.042



100.000



11.04


    

Day 2 Analysis


   

Sample Code



Conc. Obtained (mg/L)



Nominal Conc.


(mg/L)



% Recovery



Sample G1



-



-



-



Sample G2 As such formulation



89.131



100.000



89.13



Sample G2 G Filtered formulation



13.166



100.000



13.17



Sample G2 Centrifuged formulation



13.456



100.000



13.46



 


Table 8. Total fish length (cm) at the end of the test












































































































































































































































































































































































































































































































































Fish Number



Test Conc. (mg/L)



0.0



6.00



9.5



15.3



G1



G2



G3



G4



1



2



3



4



1



2



3



4



1



2



3



4



1



2



3



4



1



2.4



2.9



2.8



2.8



2.3



2.4



3.1



2.7



2.2



2.2



3.1



2.1



2.1



2.0



2.0



1.9



2



3.3



3.2



3.0



2.7



2.4



2.5



3.0



2.5



2.7



2.5



2.9



2.5



2.0



1.8



2.1



2.2



3



2.9



3.0



2.7



3.0



3.1



3.0



2.8



2.8



2.3



2.6



2.8



2.3



1.9



2.3



2.3



2.4



4



2.4



2.7



2.9



3.2



2.5



2.9



2.5



2.7



2.8



2.5



2.7



2.3



1.8



2.1



2.1



2.1



5



2.3



2.4



2.5



3.4



2.9



2.7



2.7



3.0



2.5



2.2



2.3



2.5



1.7



2.0



2.0



2.0



6



2.5



2.9



3.2



3.2



3.2



2.5



2.9



3.2



2.7



2.9



2.5



2.6



2.2



1.8



2.2



1.8



7



2.2



2.8



3.3



3.0



3.4



2.6



2.4



3.4



3.0



2.7



2.3



2.7



2.1



2.1



1.8



1.8



8



2.7



3.0



3.1



2.8



3.1



3.0



2.3



3.3



2.9



2.3



2.5



2.1



2.0



1.9



2.2



1.7



9



1.8



3.2



2.9



2.7



2.8



2.7



2.5



3.0



2.8



2.4



2.3



2.3



2.3



2.0



2.3



2.1



10



2.2



3.4



3.0



2.5



2.7



2.9



2.9



2.9



3.1



2.5



2.2



2.4



2.4



2.4



2.1



1.9



11



2.5



3.5



2.9



2.4



2.5



2.5



3.2



3.2



3.0



2.6



2.4



2.2



2.1



2.3



2.2



1.6



12



2.9



3.2



3.0



2.8



2.0



3.0



3.4



3.4



2.4



2.7



2.5



2.3



2.0



2.2



1.9



2.0



13



3.2



3.4



3.2



3.2



3.0



2.5



2.8



2.4



2.3



2.8



3.0



2.4



1.8



-



-



2.2



14



3.1



2.9



3.1



2.9



2.9



2.4



2.7



2.2



2.4



2.3



2.6



1.8



1.9



-



-



2.1



15



2.8



3.0



3.1



2.4



2.4



2.3



2.9



2.8



2.6



2.2



-



1.9



2.0



-



-



2.2



16



3.0



2.9



2.9



2.5



2.8



-



2.1



-



2.4



2.4



-



2.5



1.8



-



-



2.3



17



2.4



3.2



3.0



-



2.6



-



3.1



-



-



2.4



-



-



1.9



-



-



2.2



18



-



3.1



2.8



-



-



-



2.6



-



-



-



-



-



-



-



-



-



19



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



20



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



Individual mean



2.6



3.0



3.0



2.8



2.7



2.7



2.8



2.9



2.6



2.5



2.6



2.3



2.0



2.1



2.1



2.0



Pooled mean



2.9



2.8



2.5



2.0



Individual minimum



1.8



2.4



2.5



2.4



2.0



2.3



2.1



2.2



2.2



2.2



2.2



1.8



1.7



1.8



1.8



1.6



Pooled minimum



1.8



2.0



1.8



1.6



Individual maximum



3.3



3.5



3.3



3.4



3.4



3.0



3.4



3.4



3.1



2.9



3.1



2.7



2.4



2.4



2.3



2.4



Pooled maximum



3.5



3.4



3.1



2.4



Individual ±SD



0.41



0.27



0.19



0.31



0.37



0.24



0.33



0.36



0.28



0.21



0.28



0.24



0.19



0.20



0.15



0.22



Pooled ±SD



0.34



0.33



0.28



0.19













































































































































































































































































































































































































































































































































Fish Number



Test Conc. (mg/L)



24.4



39.1



62.5



100.0



G5



G6



G7



G8



1



2



3



4



1



2



3



4



1



2



3



4



1



2



3



4



1



1.3



1.8



1.9



1.5



1.4



1.5



1.3



1.2



1.1



1.2



1.4



1.1



0.8



-



1.0



-



2



1.2



1.6



1.9



1.9



1.5



1.3



1.5



1.5



1.0



1.1



0.8



1.0



-



-



-



-



3



1.7



1.5



2.2



1.8



1.2



1.2



1.4



1.6



1.2



1.1



1.2



1.2



-



-



-



-



4



1.8



2.3



2.4



1.8



1.6



1.3



1.3



1.1



1.2



1.2



0.9



-



-



-



-



-



5



1.5



2.1



1.8



1.7



1.5



1.5



1.2



1.2



1.3



-



0.9



-



-



-



-



-



6



1.3



2.3



1.2



1.3



1.5



1.1



1.3



1.7



1.1



-



-



-



-



-



-



-



7



1.8



2.4



1.2



1.5



1.2



1.2



1.5



1.2



1.2



-



-



-



-



-



-



-



8



2.1



1.9



1.2



1.9



1.8



1.5



1.7



1.2



1.1



-



-



-



-



-



-



-



9



2.0



2.1



1.8



2.0



1.5



1.3



1.2



1.7



-



-



-



-



-



-



-



-



10



1.8



2.4



1.3



1.8



1.3



1.1



1.4



-



-



-



-



-



-



-



-



-



11



2.0



1.8



1.8



1.9



-



1.4



1.5



-



-



-



-



-



-



-



-



-



12



1.8



1.5



2.2



-



-



1.2



-



-



-



-



-



-



-



-



-



-



13



2.0



1.7



-



-



-



-



-



-



-



-



-



-



-



-



-



-



14



1.8



1.4



-



-



-



-



-



-



-



-



-



-



-



-



-



-



15



1.6



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



16



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



17



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



18



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



19



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



20



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



Individual mean



1.7



1.9



1.7



1.7



1.5



1.3



1.4



1.4



1.2



1.2



1.1



1.1



0.8



0.0



1.0



0.0



Pooled mean



1.8



1.4



1.1



0.5



Individual minimum



1.2



1.4



1.2



1.3



1.2



1.1



1.2



1.1



1.0



1.1



0.8



1.0



0.8



0.0



1.0



0.0



Pooled minimum



1.2



1.1



0.8



0.0



Individual maximum



2.1



2.4



2.4



2.0



1.8



1.5



1.7



1.7



1.3



1.2



1.4



1.2



0.8



0.0



1.0



0.0



Pooled maximum



2.4



1.8



1.4



1.0



Individual ±SD



0.28



0.35



0.43



0.22



0.50



0.15



0.15



0.24



0.09



0.06



0.28



0.10



-



-



-



-



Pooled ±SD



0.33



0.19



0.14



0.53



 


Table 9. Fish wet weight (g) at the end of the test









































































































































































































Group



Test Conc.


 (mg/L)



Replicate



Fish wet weight


(g)



Mean Fish weight                   (g)


 
 

G1



0.0



1



1.7235



1.6351


 

2



1.5423


 

3



1.7324


 

4



1.5421


 

G2



6.0



1



1.5741



1.5034


 

2



1.6214


 

3



1.4521


 

4



1.3661


 

G3



9.5



1



1.2145



1.2551


 

2



1.4262


 

3



1.2471


 

4



1.1324


 

G4



15.3



1



1.0898



1.1273


 

2



1.1199


 

3



1.0982


 

4



1.2014


 

G5



24.4



1



1.0023



1.0495


 

2



1.1021


 

3



1.0250


 

4



1.0685


 

G6



39.1



1



0.9865



0.9676


 

2



1.0012


 

3



0.9875


 

4



0.8952


 

G7



62.5



1



0.6231



0.3294


 

2



0.2635


 

3



0.3045


 

4



0.1264


 

G8



100.0



1



0.0452



0.0494


 

2



-


 

3



0.0536


 

4



-


 
Validity criteria fulfilled:
yes
Remarks:
Dissolved oxygen was 81.2% of saturation throughout the test (> 60%). Water temperature did not differ by more than ± 1.5°C during the exposure. Overall survival of fertilized eggs in control was 91.8% and post-hatch success was 91.3% (> 70% and > 75%).
Conclusions:
Based on the results obtained under test conditions, the EC10 for hatching success was found to be 13.867 mg/L, the EC10 for larvae survival was found to be 15.259 mg/L, the EC10 based on length was 9.832 mg/L and the EC10 based on weight was 7.599 mg/L (worst case scenario), all based on nominal concentrations.
Executive summary:

A Fish, Early-life Stage Toxicity Test was performed on the test item, over a period of 30 days in semi-static conditions, according to OECD TG 210, under GLP conditions.


Based on an in-house solubility test, the test item was found to be soluble in reverse osmosis water. Based on based on the available results and from read across data, the main study was performed at the test concentrations of 6.0, 9.5, 15.3, 24.4, 39.1, 62.5 and 100.0 mg/L (nominal) and one control group.


Danio rerio (zebrafish) embryo were exposed to different test concentrations of test item in reverse osmosis water under semi-static conditions, test media was renewed once in 48 hours until the termination of the test. The test design included eight groups with four replicates per concentration and the control, containing 20 fertilized eggs per test vessel. These were observed to establish hatching success, larvae survival, fish weight, fish length, abnormal appearance and abnormal behaviour. 


A validated analytical method based on ICP-MS was used to monitor the concentration and stability of the active ingredient in the test solution. The test item was considered stable in the testing conditions after 72 hours of exposure as the measured concentrations in samples were maintained within ± 20 percent of the nominal concentrations. The test media was clear, light pink, and no particles were observed on fresh media. However, at 24 and 48 hours observations, fine particles were settled at the bottom of test vessels at the test concentrations of 62.5 and 100.0 mg/L. Therefore, additional replicates were prepared for analytical monitoring. On analytical verification at the concentration of 100 mg/L, the limit of saturation was found to be 17.37 to 20.869 mg/L. All validity criteria were met.


According to REACH guidance Chapter R.7b: Endpoint specific guidance Version 4.0 – June 2017, EC10 values are preferred as these are statistically derived from the entire dataset, and less dependent on test design considerations than the NOEC. Based on the results obtained under test conditions, the EC10 for hatching success was found to be 13.867 mg/L, the EC10 for larvae survival was found to be 15.259 mg/L, the EC10 based on length was 9.832 mg/L and the EC10 based on weight was 7.599 mg/L (worst case scenario), all based on nominal concentrations.

Description of key information

Key study. Method according to OECD 210, GLP study. The 30-day EC10 of the test item in Danio rerio (zebrafish) embryo was found to be 7.46 mg/L with 95% confidence limits 4.11 - 13.56 mg/L (nominal concentration, based on weight).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
EC10
Effect concentration:
7.46 mg/L

Additional information