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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 August 2020 - 27 January 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
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), 0.88, 1.32, 1.98, 2.96, 4.44, 6.67, and 10.0 mg/L.
- Sampling method: In order to verify the actual test concentrations, 15 mL samples were collected from low and highest test concentrations in duplicates at regular intervals. Additionally, at 0, 24, 48 and 72 hours, three samples (about 15 ml each) were collected from test media replicates in duplicates.
- Sample storage conditions before analysis: all samples were analysed on the same day of collection from the respective test vessels
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution was freshly prepared prior to exposure by weighing required quantity of test item in a beaker; then, a small volume of reconstituted water was added and mixed well using a glass rod. After complete solubility of the test item, test formulation was transferred into a measuring cylinder and the beaker was rinsed with Reconstituted water and transferred again to the measuring cylinder, rinsing process was repeated until the complete transfer of test contents. Stock solution prepared was kept on magnetic stirrer to maintain homogeneity for 30 minutes. Test medium of chosen concentration was prepared by dilution of stock solution.
- Test concentration separation factor: 1.5
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: daphnid
- Strain/clone: Daphnia magna Straus
- Age at study initiation (mean and range, SD): less than 24 hours old
- Method of breeding: 25 and 45 pregnant female Daphnids were acclimatized in the reconstituted water for 48 h prior to test during dose range finding study and main study. Daphnids had been previously maintained in reconstituted water in the test facility. Daphnids were fed with algal cells at the beginning and on Day 1 of acclimatization. Offspring produced on the first day were not used. Offspring produced on day two (less than 24 h old, derived from healthy stock showing no signs of stress) was collected and used for the test.
- Source: Environmental and Molecular Toxicology Division, Department of Zoology, Karnataka University, Dharwad, Karnataka, India.
- Feeding during test: yes
- Food type: live algal cells (Pseudokirchneriella subcapitata)
- Amount: Feeding was done within the recommended range of 0.1 to 0.2 mg C/Daphnia/day at all times.

ACCLIMATION
- Acclimation period: 48 hours
- Acclimation conditions: same as test
- Type and amount of food: Daphnids were fed with algal cells at the beginning and on Day 1 of acclimatization. Daphnids were fed with live algal cells (Pseudokirchneriella subcapitata) 0.1 to 0.2 mg of organic carbon (C) for each parent daphnid per day.
- Feeding frequency: daily
- Health during acclimation: daphnids derived from healthy stock showing no signs of stress, such as high mortality rate, discolored daphnids, presence of male and ephippia or delay in the production of the first brood, were selected for the treatment. No mortality reported.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
212 - 216 mg/L as CaCO3
Test temperature:
20.0 to 20.6ºC
pH:
7.70 to 8.39
Dissolved oxygen:
8.09 to 8.98 mg/L
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 0.88, 1.32, 1.98, 2.96, 4.44, 6.67, and 10.0 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 beakers
- Material, size, headspace, fill volume: glass, 100 mL fill volume
- Aeration: The dilution water was aerated prior to use so that the dissolved oxygen concentration reached saturation.
- Renewal rate of test solution (frequency): 48 hours, based on based on the results of stability of test item performed during analytical method validation the test media
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 100 mL of test media per daphnid.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO reconstituted water as described in the OECD 202.
- Culture medium different from test medium: no
- Intervals of water quality measurement: Total hardness of the diluent water (reconstituted water) prior to its use for exposure was analysed. Water hardness, Temperature, pH, and dissolved oxygen was recorded at daily where applicable in (fresh and spent media) in control(s) and in all the test item concentrations until termination of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours dark cycle
- Light intensity: 1000 to 1500 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Each test vessel was checked for immobilized Daphnids, mortality, and offspring daily. The offspring produced by each parent animal was removed and counted daily from the appearance of the first brood. The mortality of the parent animal was recorded daily and counted in every test vessel. Since mortality did not follow a concentration-response pattern, replicates with parental mortality were excluded from the analysis of the test result. Reproductive output per surviving parent animal (i.e., the number of living offspring produced during the test per surviving parent) was recorded. The measurement of the length of the parent animals (i.e., body length excluding the anal spine) was recorded at the end of the test. The other parameters were measured or calculated including time to production of first brood, number, and size of broods per animal, number of aborted broods, presence of male neonates, and possibly the intrinsic rate of population increase.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5
- Range finding study: A dose range finding study with 5 concentrations plus control with two replicates per dose group, under semi-static conditions. At the end of dose range finding study, reproduction of the Daphnids was assessed. The number of parents and the occurrence of offspring was recorded.
- Test concentrations: 0.01, 0.1, 1.0, 5.0 and 10.0 mg/L
- Results used to determine the conditions for the definitive study: No clinical signs of toxicity or immobility of parent daphnids was observed in the control and at the tested concentrations of 0.01, 0.1, 1.0 and 5.0 mg/L during the 21 days of exposure period. Clinical signs of lethargy and immobility in parent daphnids were observed at the parent daphnids at the tested concentration of 10.0 mg/L. The total no. of offspring produced per surviving adult was 271 in control and 273, 270, 167, 83 and 0.0 at the tested concentrations of 0.01, 0.1, 1.0, 5.0 and 10.0 mg/L, respectively.
Reduction in reproductive output was found to be -0.7, 0.4, 38.4, 69.4 and 100% at the tested concentrations of 0.01, 0.1, 1.0, 5.0 and 10.0 mg/L, respectively. The first offspring was observed after 7 days in control, similarly on day 7, at the test concentrations of 0.01 and 0.1 mg/L and on day 10 and 11 at the test concentrations of 1.0 and 5.0 mg/L. No offspring was seen and parent daphnids were observed dead on day 08 and 12 after exposure at the test concentration of 10.0 mg/L. Inhibition in growth of adult length was 0.1, -0.1, 4.5, 7.5 and 100% at the tested concentrations of 0.01, 0.1, 1.0, 5.0 and 10.0 mg/L, respectively.
Based on the results of dose range finding study, main study was performed at the test concentrations of 0.88, 1.32, 1.98, 2.96, 4.44, 6.67 and 10.0 mg/L.
Reference substance (positive control):
not required
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
2.77 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Reproductive output of offspring of F0 parent
Remarks on result:
other: 95% CL: 2.49 - 3.09 mg/L
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Reproductive output of live offspring per surviving F0 parent
Remarks on result:
other: 95% CL: 2.83 - 3.46 mg/L
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
5.55 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Reproductive output of offspring of F0 parent
Remarks on result:
other: 95% CL: 5.21 - 5.91 mg/L
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
5.46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Reproductive output of live offspring per surviving F0 parent
Remarks on result:
other: 95% CL: 5.12 - 5.83 mg/L
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
2.96 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Reproductive output of live offspring per surviving F0 parent
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
4.44 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Details on results:
- Behavioural abnormalities: none reported
- Observations on body length and weight: no abnormalities in control group. Inhibition in growth of adult length was -5.2, -2.3, 1.8, 5.8, 11.3, 26.6 and 43.3% at the tested concentrations of 0.88, 1.32, 1.98, 2.96, 4.44, 6.67 and 10.0 mg/L, respectively.
- Mortality of control: The mortality of the parent animals in control group (female Daphnia) was 0% at the end of the test (validity criteria does not exceed 20% at the end of the test).
- Other adverse effects control: no
- Immobilisation of control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no, the limit of saturation of the test item in the test media was confirmed to be 8.85 to 10.80 mg/L.
Reported statistics and error estimates:
Data were analysed by appropriate statistical methods (Finney’s probit analysis) to calculate EC10, EC20 and EC50 with confidence limits for reproductive output. NOEC/LOEC for reproductive output and daphnid length was determined using appropriate statistical test (One-way ANOVA by Dunnett t-test).

Table 1. Summary of results. 




















































































































































Response Variable



21 Day Endpoints [mg/L]



F0 adult parent survival



95%-CL



EC10



nd



EC20



nd



EC50



nd



NOEC



LOEC



Lower



-



Lower



-



Lower



-



nd



nd



Upper



-



Upper



-



Upper



-



 



 



 



 



 



 



 



 



 



 



Reproductive output of offspring of F0 parent



95%-CL



EC10



2.77



EC20



3.52



EC50



5.55



nd



nd



Lower



2.49



Lower



3.24



Lower



5.21



Upper



3.09



Upper



3.82



Upper



5.91



 



 



 



 



 



 



 



 



 



 



Reproductive output of live offspring per surviving F0 parent



95%-CL



EC10



3.13



EC20



3.79



EC50



5.46



2.96



4.44



Lower



2.83



Lower



3.50



Lower



5.12



Upper



3.46



Upper



4.11



Upper



5.83



Growth                  (Length)



95%-CL



EC10



nd



EC20



nd



EC50



nd



4.44



6.67



Lower



-



Lower



-



Lower



-



Upper



-



Upper



-



Upper



-



nd: Not determined (Data obtained is not sufficient)


 


Table 2. Surviving Adults F0 parent daphnids at the end of the test (DRF)






























































Group



Test Conc. (mg/L)



Initial No. of Parent Daphnids released



Surviving (F0) parent Daphnid at the end of the test



Percent Inhibition



Clinical signs



G1



0.0



2



2



-



N



G2



0.01



2



2



0.0



N



G3



0.1



2



2



0.0



N



G4



1.0



2



2



0.0



N



G5



5.0



2



2



0.0



N



*G6



10.0



2



0



100.0



L



N: Normal; L: Lethargy; -: Not applicable; *: Immobility observed in parent daphnids.


 


Table 3. Reproductive output of parent daphnids (DRF)
















































































Group



Test Conc. (mg/L)



Total no. of offspring produced



Total no. of offspring produced per surviving adult



Day on first brood release



F0 production rate first brood release



Total no. of offspring produced



Reduction in Reproductive Output (%)



Total no. of offspring produced



Reduction in Reproductive Output (%)



G1



0.0



271



-



271



-



7



 



G2



0.01



273



-0.7



273



-0.7



7



0.1538



G3



0.10



270



0.4



270



0.4



7



0.1538



G4



1.00



167



38.4



167



38.4



10



0.1176



G5



5.00



83



69.4



83



69.4



11



0.1053



G6



10.00



0



100.0



0



100.0



-



0.0000



-: Not applicable


 


Table 4. Comparison of response variable (DRF) 








































































Group



Test Conc. (mg/L)



Response Variable - Percent Inhibition



Adult Mortality



F0 Production Rate first brood release



Reproductive Output of live offspring per  adult



No. of live offspring per surviving F0 adult



Mean Surviving F0 adult length



G1



0.00



-



-



-



-



-



G2



0.01



0.0



0.0



-0.7



-0.7



0.1



G3



0.10



0.0



0.0



0.4



0.4



-0.1



G4



1.00



0.0



23.5



38.4



38.4



4.5



G5



5.00



0.0



31.6



69.4



69.4



7.5



G6



10.00



100.0



100.0



100.0



100



100.0



 


Table 5. Physico-chemical parameters of test medium (DRF)















































































Dissolved Oxygen (mg/L)



Group



Test Conc.



Fresh



Spent



Min



Max



Min



Max



G1



0.0



7.51



7.74



7.41



7.62



G2



0.01



7.48



7.67



7.34



7.60



G3



0.1



7.42



7.68



7.31



7.54



G4



1.0



7.42



7.74



7.22



7.62



G5



5.0



7.24



7.59



7.20



7.50



G6



10.0



7.24



7.47



7.15



7.41



Min



7.24



7.15



Max



7.74



7.62
















































































pH



Group



Test Conc.



Fresh



Spent



Min



Max



Min



Max



G1



0.0



7.17



7.37



7.10



7.31



G2



0.01



7.29



7.44



7.24



7.32



G3



0.1



7.36



7.59



7.22



7.51



G4



1.0



7.40



7.63



7.31



7.56



G5



5.0



7.39



7.86



7.31



7.80



G6



10.0



7.40



7.84



7.31



7.75



Min



7.17



7.10



Max



7.86



7.80



































Temperature (°c)



 



Fresh



Spent



Mean



20.4



20.3



±SD



0.2



0.2



Min



20.0



20.0



Max



20.7



20.7











































Measurement on



Total Hardness


[mg/L (as CaCO3)]



Acclimatization



214



Day 0 (Fresh)



216



Day 7 (Spent)



212



Day 7 (Fresh)



214



Day 14 (Spent)



216



Day 14 (Fresh)



216



Day 20 (Spent)



213



Day 20 (Fresh)



216



 


Table 6. Surviving Adults F0 parent daphnids at the end of the test (main study)














































































Group



Test Conc. (mg/L)



Initial No. of Parent Daphnids released



Surviving (F0) parent Daphnid at the end of the test



Percent Inhibition



Clinical Signs



G1



0.00



10



10



-



N



G2



0.88



10



10



0.0



N



G3



1.32



10



10



0.0



N



G4



1.98



10



10



0.0



N



G5



2.96



10



10



0.0



N



G6



4.44



10



10



0.0



 L



G7



6.67



10



9



10.0



 L, B



G8



10.0



10



 



30.0



L, B



-: Not applicable; N: Normal; L: Lethargy; B: On Bottom (localized on the bottom of the container)


 


Table 7. Reproductive output of parent daphnids (main study)




































































































Group



Test Conc. (mg/L)



Total no. of offspring produced



Total no. of offspring produced per surviving adult



Day on first brood release



F0 production rate first brood release



Total no. of offspring produced



Reduction in Reproductive Output (%)



Total no. of offspring produced



Reduction in Reproductive Output (%)



G1



0.00



1374



-



1374



-



7



0.1538



G2



0.88



1373



0.1



1373



0.1



7



0.1538



G3



1.32



1366



0.6



1366



0.6



7



0.1538



G4



1.98



1349



1.8



1349



1.8



7



0.1538



G5



2.96



1294



5.8



1294



5.8



8



0.1333



G6



4.44



993



27.7



993



27.7



9



0.1176



G7



6.67



581



57.7



573



58.3



10



0.1053



G8



10.0



80



94.2



65



95.3



14



0.0741



 


Table 8. Comparison of the response variable (main study)


























































































Group



Test Conc. (mg/L)



Response Variable - Percent Inhibition



Adult Mortality



F0 Production Rate first brood release



Reproductive Output of live offspring per  adult



No. of live offspring per surviving F0 adult



Mean Surviving F0 adult length



G1



0.00



-



-



-



-



-



G2



0.88



0.0



0.0



0.1



0.1



-5.2



G3



1.32



0.0



0.0



0.6



0.6



-2.3



G4



1.98



0.0



0.0



1.8



1.8



1.8



G5



2.96



0.0



13.3



5.8



5.8



5.8



G6



4.44



0.0



23.5



27.7



27.7



11.3



G7



6.67



10.0



31.6



57.7



58.3



26.6



G8



10.0



30.0



51.9



94.2



95.3



43.3



 


Table 9. Physico-chemical parameters of test medium (main study)































































































Dissolved Oxygen (mg/L)



Group



Test Conc.



Fresh



Spent



Min



Max



Min



Max



G1



0.0



8.24



8.98



8.17



8.90



G2



0.88



8.30



8.90



8.21



8.65



G3



1.32



8.24



8.92



8.11



8.84



G4



1.98



8.39



8.92



8.24



8.88



G5



2.96



8.29



8.92



8.17



8.85



G6



4.44



8.27



8.96



8.15



8.91



G7



6.67



8.25



8.96



8.11



8.84



G8



10.00



8.20



8.98



8.09



8.91



Min



8.20



8.09



Max



8.98



8.91
































































































pH



Group



 



Fresh



Spent



Min



Max



Min



Max



G1



0.0



8.10



8.37



8.01



8.31



G2



0.88



8.09



8.32



7.94



8.26



G3



1.32



7.90



8.36



7.76



8.30



G4



1.98



7.87



8.38



7.75



8.31



G5



2.96



7.73



8.38



7.64



8.32



G6



4.44



7.70



8.35



7.59



8.26



G7



6.67



7.80



8.36



8.00



8.32



G8



10.00



8.13



8.39



7.54



8.34



Min



7.70



7.54



Max



8.39



8.34



































Temperature (°C)


 

Fresh



Spent



Mean



20.3



20.2



±SD



0.2



0.1



Min



20.1



20.0



Max



20.6



20.5











































Measurement on



Total Hardness


[mg/L (as CaCO3)]



Acclimatization



214



Day 0 (Fresh)



216



Day 5 (Spent)



216



Day 5 (Fresh)



216



Day 14 (Spent)



212



Day 14 (Fresh)



212



Day 20 (Spent)



212



Day 20 (Fresh)



212



 


Table 10. Analytical verification of saturation concentration






























































































































Day 0 Analysis


   

Sample Code



Conc. Obtained (mg/L)



Nominal Conc.(mg/L)



% Recovery



Sample G1



-



-



-



Sample G2 As such formulation



9.458



10.000



94.58



Sample G2 G Filtered formulation



9.743



10.000



97.43



Sample G2 Centrifuged formulation



9.763



10.000



97.63


    

Day 1 Analysis


   

Sample Code



Conc. Obtained (mg/L)



Nominal Conc.(mg/L)



% Recovery



Sample G1



-



-



-



Sample G2 As such formulation



10.805



10.000



108.05



Sample G2 G Filtered formulation



10.332



10.000



103.32



Sample G2 Centrifuged formulation



9.954



10.000



99.54


    

Day 2 Analysis


   

Sample Code



Conc. Obtained (mg/L)



Nominal Conc.(mg/L)



% Recovery



Sample G1



-



-



-



Sample G2 As such formulation



9.010



10.000



90.10



Sample G2 G Filtered formulation



8.853



10.000



88.53



Sample G2 Centrifuged formulation



9.591



10.000



95.91



 


Table 11. Test concentration analysis phase report






























































































































































































































Particulars



Nominal Conc. (mg/L)



Obtained


 Conc. (mg/L)



Recovery (%)



Day - 0 Fresh (Date: 06/01/2021)



G1 - Sample



0.0



-



-



G2 - Sample



0.880



0.865



98.30



G8 - Sample



10.000



10.986



109.86



Day - 2 Spent (Date: 08/01/2021)



G1 -Sample



0.0



-



-



G2 - Sample



0.880



0.772



87.77



G8 - Sample



10.000



9.817



98.17



 



G1 - Sample



0.0



-



-



G2 - Sample



0.880



0.868



98.67



G8 - Sample



10.000



10.478



104.78



Fresh (Date: 18/01/2021)



Sample Fresh G1



0.0



-



-



Sample Fresh G2



0.880



0.840



95.44



Sample Fresh G8



10.000



10.071



100.71



Spent (Date: 18/01/2021)



Sample Spent G1



0.0



-



-



Sample Spent G2



0.880



0.828



94.05



Sample Spent G8



10.000



9.990



99.90



Fresh (Date: 20/01/2021)



Sample Fresh G1



0.0



-



-



Sample Fresh G2



0.880



0.924



105.04



Sample Fresh G8



10.000



10.244



102.44



Spent (Date: 20/01/2021)



Sample Spent G1



0.0



-



-



Sample Spent G2



0.880



0.915



104.03



Sample Spent G8



10.000



10.254



102.54



Spent (Date: 22/01/2021)



Sample G1



0.0



-



-



Sample G2



0.880



0.866



98.39



Sample G8



10.000



9.990



99.90



Day - 20 Fresh (Date: 26/01/2021)



Sample Fresh G1



0.0



-



-



Sample Fresh G2



0.880



0.954



108.46



Sample Fresh G8



10.000



11.697



116.97



Day - 21 Spent (Date: 27/01/2021)



Sample Spent G1



0.0



-



-



Sample Spent G2



0.880



0.824



93.68



Sample Spent G8



10.000



10.386



103.86



 


Table 12. Survival and reproduction of adult daphnids (main study)

























































































































































































































































































































































Group: G1, Test Conc.: 0.0 mg/L


 

Replicate



Days



1



2



3



4



5



6



7



8



9



10



1



0



0



0



0



0



0



0



0



0



0



2



0



0



0



0



0



0



0



0



0



0



3



0



0



0



0



0



0



0



0



0



0



4



0



0



0



0



0



0



0



0



0



0



5



0



0



0



0



0



0



0



0



0



0



6



0



0



0



0



0



0



0



0



0



0



7



3



1



0



2



1



0



4



1



8



0



8



5



6



3



8



8



4



3



5



9



1



9



9



7



6



9



9



9



8



9



7



7



10



7



5



10



7



12



3



2



7



10



8



11



8



11



2



8



15



5



9



8



12



9



12



13



9



8



10



16



18



13



5



15



12



13



10



7



14



12



10



13



10



10



7



14



14



12



6



19



11



8



10



14



13



9



10



15



8



10



12



9



9



12



12



18



8



11



16



17



12



14



7



11



8



8



19



11



8



17



9



14



8



8



12



9



9



16



13



9



18



15



10



9



9



10



7



8



10



16



7



19



13



8



11



5



4



12



10



11



10



16



20



10



9



11



7



5



4



13



14



8



11



21



9



11



13



5



9



9



14



11



13



8



Surviving F0 adult



1



1



1



1



1



1



1



1



1



1



No. Offspring per surviving F0 adult



148



126



140



117



139



123



137



157



156



131



Total No. of Offspring



1374.0



±SD



13.5



CV%



9.8



Total No. Offspring per surviving F0 adult



1374.0



Average No. Offspring per parent



137.4





















































































































































































































































































































































Group: G2,  Test Conc.: 0.88 mg/L



Replicate



Days



1



2



3



4



5



6



7



8



9



10



1



0



0



0



0



0



0



0



0



0



0



2



0



0



0



0



0



0



0



0



0



0



3



0



0



0



0



0



0



0



0



0



0



4



0



0



0



0



0



0



0



0



0



0



5



0



0



0



0



0



0



0



0



0



0



6



0



0



0



0



0



0



0



0



0



0



7



7



5



4



0



5



3



2



8



5



7



8



5



6



2



1



10



8



4



10



4



5



9



10



12



6



8



12



4



2



9



6



6



10



12



11



7



9



10



2



5



9



9



3



11



14



9



12



10



9



3



7



10



10



5



12



8



7



14



12



11



8



9



11



11



4



13



9



17



19



15



16



7



7



13



13



7



14



7



13



14



17



13



9



10



14



12



5



15



8



17



10



13



14



7



5



12



14



6



16



6



12



9



8



11



11



4



10



18



3



17



16



10



8



10



10



14



5



8



10



10



18



12



8



17



11



8



10



6



9



11



8



19



14



7



13



14



9



13



7



7



8



9



20



12



8



8



13



11



11



9



8



9



5



21



11



9



10



11



13



10



10



8



10



13



Surviving F0 adult



1



1



1



1



1



1



1



1



1



1



No. Offspring per surviving F0 adult



151



151



153



152



162



120



92



146



150



96



Total No. of Offspring



1373.0



±SD



25.3



Total No. Offspring per surviving F0 adult



1373



Average No. Offspring per parent



137.3





















































































































































































































































































































































Group: G3, Test Conc.: 1.32mg/L



Replicate



Days



1



2



3



4



5



6



7



8



9



10



1



0



0



0



0



0



0



0



0



0



0



2



0



0



0



0



0



0



0



0



0



0



3



0



0



0



0



0



0



0



0



0



0



4



0



0



0



0



0



0



0



0



0



0



5



0



0



0



0



0



0



0



0



0



0



6



0



0



0



0



0



0



0



0



0



0



7



3



2



1



0



0



3



0



0



0



0



8



9



9



9



7



8



2



3



7



2



9



9



10



7



10



8



13



9



8



5



9



5



10



11



9



11



11



10



11



5



10



10



7



11



14



10



8



10



14



17



7



11



17



7



12



17



11



7



15



8



13



9



13



5



8



13



8



8



9



13



9



8



10



10



6



9



14



9



9



10



18



7



11



7



5



8



10



15



7



12



13



10



13



17



8



9



9



11



16



10



17



14



11



11



12



7



7



10



14



17



12



10



16



14



10



10



11



10



14



13



18



11



8



10



13



5



8



5



13



11



15



19



8



9



7



8



17



9



7



11



10



8



20



9



7



9



9



13



7



9



14



8



9



21



13



9



10



8



11



5



2



13



5



3



Surviving F0 adult



1



1



1



1



1



1



1



1



1



1



No. Offspring per surviving F0 adult



151



137



144



155



149



142



98



138



124



128



Total No. of Offspring



1366.0



±SD



16.7



Total No. Offspring per surviving F0 adult



1366



Average No. Offspring per parent



136.6





















































































































































































































































































































































Group: G4, Test Conc.: 1.98 mg/L



Replicate



Days



1



2



3



4



5



6



7



8



9



10



1



0



0



0



0



0



0



0



0



0



0



2



0



0



0



0



0



0



0



0



0



0



3



0



0



0



0



0



0



0



0



0



0



4



0



0



0



0



0



0



0



0



0



0



5



0



0



0



0



0



0



0



0



0



0



6



0



0



0



0



0



0



0



0



0



0



7



3



0



2



1



0



8



4



1



2



4



8



2



3



8



10



8



9



6



1



3



10



9



9



5



10



9



9



10



9



9



9



13



10



7



9



11



10



10



11



10



2



10



11



11



10



10



19



11



13



10



5



7



11



8



12



11



11



10



11



10



8



8



14



4



9



13



15



14



17



13



11



9



4



8



8



10



14



13



18



16



15



14



7



7



6



9



11



15



17



19



8



12



15



11



8



9



11



14



16



3



8



9



11



13



12



9



7



10



12



17



9



5



10



14



8



14



10



8



8



8



18



8



7



11



2



10



10



11



5



4



17



19



7



9



14



8



11



8



14



4



5



8



20



11



5



10



9



7



9



13



9



7



9



21



13



6



9



10



9



10



8



1



8



8



Surviving F0 adult



1



1



1



1



1



1



1



1



1



1



No. Offspring per surviving F0 adult



138



129



164



146



148



146



126



91



109



152



Total No. of Offspring



1349.0



±SD



21.8



Total No. Offspring per surviving F0 adult



1349



Average No. Offspring per parent



134.9





















































































































































































































































































































































Group: G5, Test Conc.: 2.96 mg/L



Replicate



Days



1



2



3



4



5



6



7



8



9



10



1



0



0



0



0



0



0



0



0



0



0



2



0



0



0



0



0



0



0



0



0



0



3



0



0



0



0



0



0



0



0



0



0



4



0



0



0



0



0



0



0



0



0



0



5



0



0



0



0



0



0



0



0



0



0



6



0



0



0



0



0



0



0



0



0



0



7



0



0



0



0



0



0



0



0



0



0



8



0



0



0



0



0



0



0



2



0



0



9



5



5



3



0



12



9



8



10



5



0



10



5



9



5



0



15



7



9



7



18



5



11



6



14



7



0



14



11



10



14



10



9



12



8



11



9



1



10



14



11



15



9



7



13



12



19



10



11



13



10



12



18



14



8



14



10



15



13



15



15



8



14



10



9



10



15



9



11



19



13



11



6



17



11



11



9



16



8



10



9



12



14



7



11



11



14



5



17



4



18



14



10



12



18



13



9



18



7



18



6



8



10



9



18



9



11



11



10



6



19



5



9



10



10



13



10



2



13



8



5



20



7



10



17



15



14



9



6



10



7



6



21



9



9



12



9



10



8



7



13



9



8



Surviving F0 adult



1



1



1



1



1



1



1



1



1



1



No. Offspring per surviving F0 adult



94



148



138



105



171



126



131



154



142



85



Total No. of Offspring



1294.0



±SD



27.4



Total No. Offspring per surviving F0 adult



1294



Average No. Offspring per parent



129.4





















































































































































































































































































































































Group: G6, Test Conc.: 4.44 mg/L



Replicate



Days



1



2



3



4



5



6



7



8



9



10



1



 0



0



0



0



0



0



0



0



0



0



2



0



0



0



0



0



0



0



0



0



0



3



0



0



0



0



0



0



0



0



0



0



4



0



0



0



0



0



0



0



0



0



0



5



0



0



0



0



0



0



0



0



0



0



6



0



0



0



0



0



0



0



0



0



0



7



0



0



0



0



0



0



0



0



0



0



8



0



0



0



0



0



0



0



0



0



0



9



1



3



8



3



0



8



8



0



0



0



10



11



8



9



2



7



9



12



1



7



0



11



8



10



5



4



8



11



10



5



8



5



12



7



12



8



5



8



10



12



9



9



3



13



9



8



9



6



9



5



9



10



11



5



14



4



7



10



7



8



7



8



11



10



9



15



3



10



11



9



14



9



10



9



13



1



16



2



12



8



6



12



5



11



7



14



7



17



8



8



7



5



11



7



13



5



10



5



18



4



6



8



4



10



9



18



9



8



2



19



9



7



6



7



9



10



10



10



9



6



20



7



5



5



9



8



11



17



11



11



3



21



7



6



4



1



7



14



13



12



8



5



Surviving F0 adult



1



1



1



1



1



1



1



1



1



1



No. Offspring per surviving F0 adult



80



102



98



68



111



115



151



99



118



51



Total No. of Offspring



993.0



±SD



28.1



Total No. Offspring per surviving F0 adult



993.0



Average No. Offspring per parent



99.3





















































































































































































































































































































































Group: G7, Test Conc.: 6.67 mg/L



Replicate



Days



1



2



3



4



5



6



7



8



9



10



1



0



0



0



0



0



0



0



0



0



0



2



0



0



0



0



0



0



0



0



0



0



3



0



0



0



0



0



0



0



0



0



0



4



0



0



0



0



0



0



0



0



0



0



5



0



0



0



0



0



0



0



0



0



0



6



0



0



0



0



0



0



0



0



0



0



7



0



0



0



0



0



0



0



0



0



0



8



0



0



0



0



0



0



0



0



0



0



9



0



0



0



0



0



0



0



0



0



0



10



0



3



2



4



3



8



5



8



0



0



11



3



2



2



5



5



0



2



5



5



2



12



2



8



6



9



6



0



9



6



0



4



13



4



9



9



7



8



1



8



4



0



2



14



0



5



4



0



10



7



7



2



1



-



15



0



4



8



6



1



9



5



6



8



-



16



2



3



7



4



8



2



3



9



7



-



17



4



8



9



1



9



10



6



5



5



-



18



8



9



3



0



5



8



5



1



10



-



19



9



5



10



9



7



9



4



3



10



-



20



2



7



12



5



6



13



8



4



7



-



21



1



5



8



0



5



10



9



5



8



-



Surviving F0 adult



1



1



1



1



1



1



1



1



1



D



No. Offspring per surviving F0 adult



35



68



80



50



73



77



71



58



61



8



Total No. of Offspring



581.0



±SD



14.4



Total No. Offspring per surviving F0 adult



573



Average No. Offspring per parent



63.7



 


Table 13. Individual length of adult daphnids at termination (main study)



















































































































































Group



Test Conc. (mg/L)



Replicates



Mean Length



1



2



3



4



5



6



7



8



9



10



Length of Parent Daphnids Measured (µm)



G1



0.00



2512.046



2461.324



2244.303



1257.348



2032.155



1920.385



2192.443



2212.537



2196.196



2219.814



2124.855



G2



0.88



2045.735



2450.471



2439.11



2607.740



2163.172



1931.967



2051.846



2052.012



2181.086



2420.215



2234.335



G3



1.32



2164.855



2120.341



2245.114



2170.158



2197.93



2278.396



2139.08



2201.918



2270.112



1946.688



2173.459



G4



1.98



2001.474



1932.98



1990.041



2193.778



1939.655



2006.656



2448.713



2246.641



1943.854



2161.186



2086.498



G5



2.96



2104.927



2026.338



2001.51



2247.424



1946.787



2015.621



1965.288



1956.139



1995.983



1756.732



2001.675



G6



4.44



2053.277



1832.697



2243.831



1395.772



1957.517



2046.183



1240.681



2034.268



2195.403



1848.297



1884.793



G7



6.67



1907.912



1870.858



1803.625



1181.922



1181.94



1437.941



2102.863



1376.396



1181.142



-



1560.511



G8



10.0



1062.490



1155.197



1023.794



1412.021



1409.039



1165.659



1198.182



-



-



-



1203.769



 

Validity criteria fulfilled:
yes
Remarks:
Mortality of parent animals in controls was 0% at the end of the test (< 20%). Mean no. of offspring per parent animal surviving was 137.4 (>60). CV% around mean no. of offspring was 0.4 (≤ 25%). Dissolved O2 was 8.09 (> 3 mg/L).
Conclusions:
Based on the results obtained under test conditions, the EC10 for reproductive output of offspring of F0 was 2.77 mg/L (95% CL 2.49 - 3.09), and the EC10 for reproductive output of live offspring per surviving F0 parent was 3.13 mg/L (95% CL 2.83 - 3.46); the NOEC for daphnid growth was 4.44 mg/L, all based on nominal concentrations.
Executive summary:

A Daphnia magna reproduction test was performed on the test item, over a period of 21 days in semi-static conditions, according to OECD TG 211, under GLP conditions. 


Based on an in-house solubility test, the test item was found to be soluble in reconstituted water. Based on the performed preliminary range-finding test performed at 0 (control), 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, the main study was performed at the test concentrations of 0.88, 1.32, 1.98, 2.96, 4.44, 6.67, and 10.0 mg/L (nominal) and one control group. 24-hour old daphnids were exposed to the test item in ISO reconstituted water under semi-static conditions, test media was renewed once in 48 hours until the termination of the test. Daphnids were observed for signs of toxicity, immobility of parent daphnids, and reproductive output of the surviving parent daphnids. The test design included ten replicates per concentration and for the untreated control, containing one parent animal per test vessel. 


Each test vessel was checked for immobilized Daphnids, mortality, and offspring daily. The offspring produced by each parent animal was removed and counted daily from the appearance of the first brood. The mortality of the parent animal was recorded daily and counted in every test vessel. Reproductive output per surviving parent animal (i.e., the number of living offspring produced during the test per surviving parent) was recorded. The measurement of the length of the parent animals (i.e., body length excluding the anal spine) was recorded at the end of the test. The other parameters were measured or calculated including time to production of first brood, number, and size of broods per animal, number of aborted broods, presence of male neonates, and possibly the intrinsic rate of population increase.


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 blue, and no particles were observed at the bottom of test vessels at 24- and 48-hour observation, at all tested concentrations. Additionally, the limit of saturation of the test item in the test media was confirmed to be 8.85 to 10.80 mg/L in a separate test. All validity criteria were met.


After 21 days of exposure, clinical signs of lethargy, daphnids localized on the bottom of the container, and 10 and 30.0% mobility inhibition were observed at the tested concentrations of 6.67 and 10.0 mg/L. Reduction in reproductive output per surviving adult was found to be 0.1, 0.6, 1.8, 5.8, 27.7, 57.7 and 94.2% at the tested concentrations of 0.88, 1.32, 1.98, 2.96, 4.44, 6.67 and 10.0 mg/L, respectively. Inhibition in growth of adult length was -5.2, -2.3, 1.8, 5.8, 11.3, 26.6 and 43.3% at the tested concentrations of 0.88, 1.32, 1.98, 2.96, 4.44, 6.67 and 10.0 mg/L, respectively. Since mortality did not follow a concentration-response pattern, replicates with parental mortality were excluded from the analysis of the test result.


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 reproductive output of offspring of F0 was 2.77 mg/L (95% CL 2.49 - 3.09), and the EC10 for reproductive output of live offspring per surviving F0 parent was 3.13 mg/L (95% CL 2.83 - 3.46); the NOEC for daphnid growth was 4.44 mg/L, all based on nominal concentrations.

Description of key information

Key study. Method according to OECD 211, GLP study. The 21-day EC10 of the test item in Daphnia Magna was found to be 2.77 mg/L with 95% confidence limits 2.49 - 3.09 mg/L (nominal concentration, based on reproductive performance). 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC10
Effect concentration:
2.77 mg/L

Additional information