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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:
2020-09-15 to 2020-10-07
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
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
At the start of the exposure intervals, on day 0, 7, 14 and 16, samples of the fresh media were taken after preparation of all test item concentrations and analyzed.
At the end of the exposure intervals, on day 1, 8, 15 and 17 (24 hours), samples of the old media were taken directly from the test vessels
Vehicle:
no
Details on test solutions:
A stock solution of 200 mg dispersion/L of the test item was prepared with the culture medium for each exposure interval. An appropriate amount of the test item was weighed out and transferred into a measuring flask with an appropriate amount of dilution water. The stock solution was shaken manually at room temperature. The stock solution was used for the preparation of the test concentrations by dilution with the dilution water.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: STRAUS
- Age at study initiation: Less than 24 hours old daphnids from a healthy stock
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Breeder: Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Culture stock: Reared in glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20±2 °C, 16 hours illumination, light intensity of max. 1500 lx
- Culture medium: Elendt M4, according to OECD 211, Annex 2, is used.
- Feeding of the culture stocks: The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae are cultured at the test facility.


ACCLIMATION
- Acclimation period: Acclimatization was not necessary, because the composition of the dilution water was equivalent to the culture medium
- Type and amount of food:
Daily; Pseudokirchneriella subcapitata (0.566 – 0.863 mL) and Desmodesmus subspicatus (0.502 - 0.746 mL) suspension was provided as food corresponding to max. 0.2 mg C per Daphnia and day. The density of the algae suspension varied, but it was the same for all test groups on each feeding day.



Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period were used for the test. No first brood progeny was used for the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
control: 244 - 326 mg/L as CaCO3
Highest concentration level: 239 - 314 mg/L as CaCO3
Test temperature:
The temperature was in the range of 18 – 22 °C and did not vary by more than 2 °C.
Control: 20.2 - 21.0°C
Highest concentration level: 20.2 - 20.9°C
pH:
control: 7.95 - 8.40
Highest concentration level: 7.52 - 8.37
Dissolved oxygen:
control: 6.88 - 9.61 mg/L
Highest concentration level: 7.27 - 8.90 mg/L
Nominal and measured concentrations:
2.00 - 6.32 - 20.0 - 63.2 - 200 mg dispersion/L (separation factor: √10), corresponding to 0.208 - 0.658 - 2.08 - 6.58 - 20.8 mg test item/L (dry content 10.4%) and the time weighted mean measured concentrations 0.0935 - 0.263 - 0.956 - 2.38 - 9.29 mg test item/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity, loosely covered with watch glasses
- Test volume: 50 mL
- Aeration: Test vessels were not aerated during the test.
- Renewal rate of test solution: daily renewal
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
Elendt M4, according to OECD 211, Annex 2

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality of parental daphnids: once a day at least at the same time as the offspring was counted. Dead daphnids were removed as soon as they were detected.

Neonates: First appearance of juveniles was checked daily. The number of neonates (alive and dead progeny) was counted and dates of release were recorded once a day from the first day of appearance of neonates until the end of the exposure. The neonates (alive and dead) were removed after counting and before addition of algae to prevent them from consuming food intended for the adults. The number of aborted eggs or dead offspring and dates of observation were recorded.

Additional observations /Condition of parental daphnids: Abnormalities (e.g. abnormal swimming behavior, number of males and winter eggs) were observed and recorded on each day of observation.

Total body length / Mean dry weight of parental daphnids: At the end of the test, the total length excluding the anal spine of each survived parental daphnid and the mean dry weight of the survived parental daphnids of all test concentrations and the control were determined.

Additional endpoints: The time to first brood, the intrinsic rate of population increase and the number and size of first brood per animal were reported, but not used for endpoint calculations.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: sqrt(10)

- Range finding study
- Test concentrations: of 0.100, 1.00 and 10.0 mg test item dispersion/L; five daphnids, one daphnia per replicate, were exposed to each concentration level and the control; shortened reproduction test (at least until observation of the first brood in all test groups) under semi-static conditions with a renewal of the test solutions every two or three days over a period of 9 days.
- Results used to determine the conditions for the definitive study:
no adult mortality, no difference in number of offspring after 9 days per parent daphnid, first appearance of living juveniles after 7-8 d in all groups

Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.268 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.984 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: adult mortality
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.346 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% ci: 0.150 – 0.797 mg/L
Details on results:
The test item was dispersed into the dilution water. The freshly prepared stock solution (200 mg dispersion/L) for each day was homogenous and slightly milky.

The average number of living juveniles at the end of the test after 21 days per survived parental daphnid was 95.6. in the control group. Thus, the validity criterion for the reproduction of a mean of ≥ 60 living offspring per survived parent animal in the control was met.
No parental daphnids were excluded due to accidental or inadvertent mortality from the evaluation of the reproductive output. Since Cochran-Armitage test procedure revealed a significant increasing trend (p ≤ 0.05) in adult mortality, the evaluation was done based on cumulative offspring per introduced parent. A statistically significant reduction of the reproductive output in comparison to the reproductive output in the control was determined at the time weighted mean measured concentration levels 0.984 - 2.41 and 9.29 mg test item/L and the corresponding nominal test item concentration 2.08 – 6.58 and 20.8 mg test item/L (dry content).
The determination of the EC10 / 20 / 50-value and their 95% confidence limits was carried out by probit analysis. The EC50 Reproduction was 0.792 mg/L (95% confidence limits: 0.303 – 2.118 mg/L), the LOECReproduction was 0.984 mg/L and the NOECReproduction was 0.268 mg/L based on the time weighted mean measured test item concentrations.

Accidental and inadvertent mortality as defined in the OECD test guideline was not observed. The test item induced statistically significant adult mortality after 21 days.
The mortality rate of the introduced adults was 100% in the time weighted mean measured concentration level 2.41 and 9.29 mg test item/L, 20% in the time weighted mean measured concentration level 0.984 mg test item/L, 0% in the time weighted mean measured concentration level 0.268 mg test item/L and 10% in the time weighted mean measured concentration level 0.0935 mg test item/L. In the control 20% mortality of the introduced adults was observed until the end of the test (21 days). The Cochran-Armitage test procedure revealed a significant increasing trend (p ≤ 0.05) in adult mortality. A statistically significant dose-response was detected. The determination of the LC/LL50-value was carried out by probit analysis. The confidence limits were estimated from the observed data. The LC50 was 1.02 mg/L (confidence limits: 0.984 - 2.41 mg/L), the LOECAdult mortality was 2.41 mg/L and the NOECAdult mortality was 0.984 mg/L based on the time weighted mean measured test item concentrations.

The intrinsic rates of natural increase (IR) of the survived parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance.

The first appearance of living juveniles was observed between days 7 and 11 at the introduced parental daphnids of the control and the concentration levels 0.0935 to 0.984 mg/L. In the two highest concentration levels no living juveniles were observed.

The mean values of the body length (excluding the anal spine) of the survived parental daphnids in the tested concentration levels 0.0935 to 0.984 mg/L were in the range of 3.5 to 4.5 mm per daphnid and 4.5 mm per daphnid in the control group. For this parameter, a decrease with increasing test item concentrations was observed.
The mean dry weight of the survived parental daphnids was in the range of 0.10 to 0.61 mg per daphnid in the concentration levels 0.0935 to 0.984 mg/L and 0.46 mg per daphnid in the control.

No males were observed in the control or in the test groups during the test.
No ephippia were observed in the control or in the test groups during the test.
Reported statistics and error estimates:
The NOEC and LOEC for reproduction and adult mortality were determined with ToxRat Professional as specified below. Significant differences of reproduction were determined in comparison to the control using statistical standard procedures as normality test (Shapiro-Wilk’s test), variance homogeneity test (Levene’s test), Multiple Sequentially-rejective Median (2x2-Table) Test After Bonferroni-Holm for reproduction and Step-down Cochran-Armitage Test Procedure for adult mortality.
Prior to running the Multiple Sequentially-rejective Median (2x2-Table) Test as well as the Step-down Cochran-Armitage Test, a normality test and a variance homogeneity test were performed. Before the Step-down Cochran-Armitage Test, a monotonicity test was performed additionally. P values of normality and variance homogeneity test were 0.01. The -value (acceptable probability of incorrectly concluding that there is a difference) was  = 0.05 for Multiple Sequentially-rejective Median (2x2-Table) test and the Step-down Cochran-Armitage Test.
For effects on reproduction, the juveniles per introduced parent at the start of the test excluding accidental and inadvertent mortality (reproductive output) were counted. Since no accidental or inadvertent mortality was observed, no parental daphnids were excluded from the evaluation of the reproductive output.

The EC10 / 20 / 50 -values for the reproductive output with 95% confidence limits were calculated by probit analysis with ToxRat Professional.
The LC10 / 20 / 50-value for the adult mortality were calculated by probit analysis with ToxRat Professional. The confidence limits could not calculated with ToxRat Professional. Therefore, the confidence limits were estimated empirically from the observed adult mortality.

The intrinsic rate of natural increase (IR) was calculated for the survived parental daphnids with the following EULER-LOTKA-equation with the software ToxRat Professional.

Validity Criteria


Mortality of parent animal in the control group should not exceed 20% at the end of the test. The actual mortality of parent animals in the control group was 20%.


The mean number of living offspring produced per survived parent animal at the end of the test was 95.6 in the control group (required: at least 60 juveniles per survived parental daphnid).


Effects on Reproduction for all Introduced Parents

























































 


Nominal test item concentration


 


[mg test item/L]



Time weighted mean measured test item concentration


[mg/L]



Mean number of offspring
per introduced parental daphnid



Mean



SD



CV



20.8



9.29



0*



-



-



  6.58



2.41



0*



-



-



  2.08



0.984



31.5*



15.6



49.6



  0.658



0.268



75.4



14.4



19.1



  0.208



0.0935



85.4



26.2



30.6



Control



79.7



35.5



44.6



* statistically significant different to the control


 


Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure (n = 10)

























































Nominal test item concentrations


[mg/L]


(dry content)



Time weighted mean measured test item concentration


[mg/L]



Adult Mortality [%]



7 days



14 days



21 days



20.8



9.29



100



100



100



  6.58



2.41



0



40



100



  2.08



0.984



0



0



20



  0.658



0.268



0



0



0



  0.208



0.0935



0



10



10



Control



0



10



20



 


Intrinsic Rates of Natural Increase

















































Nominal test item


concentration


 


[mg test item/L]



Time weighted mean


measured test item concentration


[mg/L]



Mean IR



CV



MV ± SD



[%]



20.8



9.29



No survived parental daphnids



  6.58



2.41



No survived parental daphnids



  2.08



0.984



0.297 ± 0.0097



3.3



  0.658



0.268



0.344 ± 0.0204



5.9



  0.208



0.0935



0.337 ± 0.0138



4.1



Control



0.336 ± 0.0351



10.4



 


First Appearance of Living Juveniles in the Individual Groups





















































































































 


Nominal test item concentration


 


[mg test item/L]



Time weighted mean measured test item concentration


[mg/L]



Day of first appearance of living juveniles at the introduced parental daphnids in replicate no.



First



appearance



1



2



3



4



5



6



7



8



9



10



mean day



20.8



9.29



-



-



-



-



-



-



-



-



-



-



-



  6.58



2.41



-



-



-



-



-



-



-



-



-



-



-



  2.08



0.984



8



8



8



8



8



8



8



8



8



8



8.0



  0.658



0.268



8



8



8



8



8



8



8



8



8



8



8.0



  0.208



0.0935



8



8



8



8



8



8



8



8



8



8



8.0



Control



7



11



9



8



8



8



8



8



8



8



8.3




  • = no living juveniles were observed


 


Total Body Length and Dry Weight of the Survived Parental Daphnids








































































































































Time weighted mean measured test item concentration


[mg/L]



Total length of the survived parent animals [mm]



MV



N



Dry weight


[mg]



Replicate no.



 



 



1



2



3



4



5



6



7



8



9



10



[mm]



 



S



MV



9.29



-



-



-



-



-



-



-



-



-



-



-



-



-



-



2.41



-



-



-



-



-



-



-



-



-



-



-



-



-



-



0.984



3.5



3.5



3.5



3.5



3.5



-



3.0



-



3.5



4.0



3.5



8



0.8



0.10



0.268



4.5



4.5



4.0



4.0



4.0



4.5



4.5



4.0



4.0



4.0



4.2



10



4.6



0.46



0.0935



4.5



4.5



-



4.0



5.0



4.5



5.0



4.5



4.5



4.0



4.5



9



5.5



0.61



Control



4.5



4.5



4.5



-



4.5



4.5



4.5



-



4.5



4.5



4.5



8



3.7



0.46




  • N = number of survived parental daphnids

  • -- = not applicable, due to mortality of parental daphnid(s)


 


 


 


Observations at Test and Control Groups


Observations in the Control










































































































































































































































































































































Day



1



2



3



4



5



6



7



8



9



10



11



12



13



14



15



16



17



18



19



20



21



 



Replicate No.



Number of Juveniles



total



1



-



-



-



-



-



-



8



-



-



11



-



-



24



-



-



28



-



-



-



25



-



96



2



-



-



-



-



-



-



-



-



-



-



15



-



-



22



-



-



25



-



-



31



-



93



3



-



-



-



-



-



-



-



-



5



-



-



19



-



-



31



-



-



33



-



-



-



88



4



-



-



-



-



-



-



-



3



-



-



15



-



-



10



M



 



28



5



-



-



-



-



-



-



-



7



-



-



19



-



-



21



-



-



38



-



-



31



-



116



6



-



-



-



-



-



-



-



9



-



-



13



-



-



30



-



-



24



-



-



13



7



96



7



-



-



-



-



-



-



-



4



-



-



7



-



-



22



-



-



25



-



-



21



-



79



8



-



-



-



-



-



-



-



4



-



-



M



 



4



9



-



-



-



-



-



-



-



7



-



-



15



-



-



27



-



-



27



-



-



31



-



107



10



-



-



-



-



-



-



-



8



-



-



18



-



-



23



-



-



23



-



-



18



-



90



Based on all parental daphnids introduced at the start of the exposure:



797



Based on parental daphnids survived until the end of the test:



765



Mortality of Parental (N)



0



0



0



0



0



0



0



0



0



0



1



1



1



1



2



2



2



2



2



2



2



2



Mortality of Parental [%]



0



0



0



0



0



0



0



0



0



0



10



10



10



10



20



20



20



20



20



20



20



20



 


-      = no juveniles observed


M    = dead parental daphnid


 


 


Observations in the Time Weighted Mean Measured Test Item Concentration 0.0935 mg/L















































































































































































































































































































































Day



1



2



3



4



5



6



7



8



9



10



11



12



13



14



15



16



17



18



19



20



21



 



Replicate No.



Number of Juveniles



total



1



-



-



-



-



-



-



-



7



-



-



13



1



-



16



-



-



23



-



-



25



-



85



2



-



-



-



-



-



-



-



5



-



-



16



-



-



8



-



-



25



-



-



32



-



86



3



-



-



-



-



-



-



-



8



-



-



M



 



18



4



-



-



-



-



-



-



-



11



-



-



13



-



-



21



-



-



16



-



-



17



-



78



5



-



-



-



-



-



-



-



3



-



-



16



-



-



25



-



-



30



-



-



31



-



105



6



-



-



-



-



-



-



-



7



-



-



13



-



-



22



-



-



26



-



-



23



-



91



7



-



-



-



-



-



-



-



4



-



-



17



-



-



19



-



7



21



-



-



30



-



98



8



-



-



-



-



-



-



-



1



-



-



19



-



-



23



-



-



30



-



-



31



-



104



9



-



-



-



-



-



-



-



5



-



-



13



-



-



27



-



-



32



-



-



33



-



110



10



-



-



-



-



-



-



-



4



-



-



16



-



-



19



-



-



25



-



-



15



-



79



Based on all parental daphnids introduced at the start of the exposure:



854



Based on parental daphnids survived until the end of the test:



836



Mortality of Parental (N)



0



0



0



0



0



0



0



0



0



0



1



1



1



1



1



1



1



1



1



1



1



1



Mortality of Parental [%]



0



0



0



0



0



0



0



0



0



0



10



10



10



10



10



10



10



10



10



10



10



10



 


-      = no juveniles observed


M    = dead parental daphnid


 


 


 


Observations in the Time Weighted Mean Measured Test Item Concentration 0.268 mg/L
























































































































































































































































































































































Day



1



2



3



4



5



6



7



8



9



10



11



12



13



14



15



16



17



18



19



20



21



 



Replicate No.



Number of Juveniles



total



1



-



-



-



-



-



-



-



6



-



-



15



-



-



20



-



-



21



-



-



23



-



85



2



-



-



-



-



-



-



-



6



-



-



17



-



-



25



-



-



25



-



-



30



-



103



3



-



-



-



-



-



-



-



7



-



-



9



-



-



10



-



-



14



-



-



25



-



65



4



-



-



-



-



-



-



-



5



-



-



17



-



-



17



-



-



10



-



-



20



-



69



5



-



-



-



-



-



-



-



4



-



-



9



-



21



-



-



-



8



-



-



14



-



56



6



-



-



-



-



-



-



-



9



-



-



14



2



-



12



-



-



15



-



-



24



-



76



7



-



-



-



-



-



-



-



8



-



1



14



1



20



-



-



-



17



-



-



23



-



84



8



-



-



-



-



-



-



-



6



-



-



19



-



1



17



-



-



18



-



-



19



-



80



9



-



-



-



-



-



-



-



7



-



-



10



-



2



18



-



-



8



-



-



11



-



56



10



-



-



-



-



-



-



-



13



-



-



16



-



-



17



-



-



15



-



-



19



-



80



Based on all parental daphnids introduced at the start of the exposure:



754



Based on parental daphnids survived until the end of the test:



754



Mortality of Parental (N)



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



Mortality of Parental [%]



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



 


-      = no juveniles observed


 


 


 


Observations in the Time Weighted Mean Measured Test Item Concentration 0.984 mg/L
















































































































































































































































































































































Day



1



2



3



4



5



6



7



8



9



10



11



12



13



14



15



16



17



18



19



20



21



 



Replicate No.



Number of Juveniles



total



1



-



-



-



-



-



-



-



7



-



-



6



-1



-1



-1



-1



-1



-1



-



61



-1



-



19



2



-



-



-



-



-



-



-



5



-



-



9



-



-



-



-



-



13



-



-



13



-



40



3



-



-



-



-



-



-



-



6



-



-



10



-



-



8



-



-



8



-



-



15



-



47



4



-



-



-



-



-



-



-



6



-



-



7



-



-



-



-



-



11



-



-



12



-



36



5



-



-



-



-



-



-



-



3



-



-



12



-



-



7



-



-



5



-



-



10



-



37



6



-



-



-



-



-



-



-



5



-



-



9



-



-



-



-



M



 



14



7



-



-



-



-



-



-



-



7



-



-



4



-1



-1



-1



-1



-1



-1



-1



-1



-1



81



19



8



-



-



-



-



-



-



-



6



-



-



7



-



-



3



-



M



 



16



9



-



-



-



-



-



-



-



7



-



-



10



-



-



-



-



-



-



6



2



-



-



25



10



-



-



-



-



-



-



-



9



-



-



16



-



-



4



-



-



12



-



-



21



-



62



Based on all parental daphnids introduced at the start of the exposure:



315



Based on parental daphnids survived until the end of the test:



285



Mortality of Parental (N)



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



2



2



2



2



2



2



2



Mortality of Parental [%]



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



20



20



20



20



20



20



20



 


-      = no juveniles observed


1)     = pale parental daphnid


M    = dead parental daphnid


 


 


Observations in the Time Weighted Mean Measured Test Item Concentration 2.41 mg/L
























































































































































































































































































































































Day



1



2



3



4



5



6



7



8



9



10



11



12



13



14



15



16



17



18



19



20



21



 



Replicate No.



Number of Juveniles



total



1



-



-



-



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



M



 



 



 



 



 



0



2



-



-



-



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



M



 



 



 



 



 



 



0



3



-



-



-



-1



-1



-1



-1



-1



-1



-1



-1



M



 



 



 



 



 



 



 



 



 



0



4



-



-



-



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



M



 



 



 



0



5



-



-



-



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



M



 



 



 



 



 



 



0



6



-



-



-



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



M



 



 



 



 



 



 



0



7



-



-



-



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



M



 



 



 



 



 



 



 



0



8



-



-



-



-1



-1



-1



-1



-1



-1



-1



-1



-1



M



 



 



 



 



 



 



 



 



0



9



-



-



-



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



-1



M



 



 



 



0



10



-



-



-



-1



-1



-1



-1



-1



-1



-1



-1



M



 



 



 



 



 



 



 



 



 



0



Based on all parental daphnids introduced at the start of the exposure:



0



Based on parental daphnids survived until the end of the test:



0



Mortality of Parental (N)



0



0



0



0



0



0



0



0



0



0



0



2



3



4



7



8



8



10



10



10



10



10



Mortality of Parental [%]



0



0



0



0



0



0



0



0



0



0



0



20



30



40



70



80



80



100



100



100



100



100



 


-      = no juveniles observed


1)     = pale parental daphnid


M    = dead parental daphnid


 


 


Observations in the Time Weighted Mean Measured Test Item Concentration 9.29 mg/L











































































































































































Day



1



2



3



4



5



6



7



8



9



10



11



12



13



14



15



16



17



18



19



20



21



 



Replicate No.



Number of Juveniles



total



1



-



M



 



0



2



-



M



 



0



3



-



-



M



 



0



4



-



M



 



0



5



-



-



M



 



0



6



-



-



M



 



0



7



-



-



M



 



0



8



-



-



M



 



0



9



-



-



M



 



0



10



-



-



M



 



0



Based on all parental daphnids introduced at the start of the exposure:



0



Based on parental daphnids survived until the end of the test:



0



Mortality of Parental (N)



0



3



10



10



10



10



10



10



10



10



10



10



10



10



10



10



10



10



10



10



10



10



Mortality of Parental [%]



0



30



100



100



100



100



100



100



100



100



100



100



100



100



100



100



100



100



100



100



100



100



 


-      = no juveniles observed


M    = dead parental daphnid
1)       = pale parental daphnid


 


 


 

Validity criteria fulfilled:
yes
Conclusions:
The effects on reproduction were evaluated based on the reproduction per introduced parent animal, since this parameter is the ecologically most relevant response variable and is required by OECD test guideline 211 (2012), when a significant trend in mortality is detected.
The overall effect threshold for effects of the test item under the test conditions was 0.268 mg/L (NOEC) and 0.984 mg/L (LOEC) based on the time weighted mean measured concentrations of the test item. The EC50 for reproduction was determined to be 0.792 (0.303 – 2.118) mg/L based on the time weighted mean measured concentrations of the test item.
The overall effect threshold for effects of the test item under the test conditions was 0.658 mg/L (NOEC) and 2.08 mg/L (LOEC) based on the nominal concentration of the test item .
Executive summary:

A Daphnia magna reproduction test (semi-static, 21 d) of partially unsaturated TEA-Esterquat was conducted at the test facility according to OECD 211 (2012). The test species was Daphnia magna STRAUS. Ten daphnids, held individually, were used per concentration level and control. At test start the daphnids were < 24 hours old. The study was carried out under semi-static conditions with a daily renewal. The aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.


Nominal concentrations of the test item were selected based on the results of a preliminary range finding test (non-GLP) as follows: 2.00 - 6.32 - 20.0 - 63.2 - 200 mg dispersion/L (separation factor √10), corresponding to 0.208 – 0.658 – 2.08 – 6.58 – 20.8 mg test item/L (dry content of the suspension: 10.4%) and the time weighted mean measured concentrations 0.0935 - 0.268 - 0.984 - 2.41 - 9.29 mg test item/L.


A stock solution with a nominal concentration of 200 mg dispersion/L was prepared as specified in section 5.2 and was used for the preparation of the concentration levels in the range of 2.00 - 200 mg dispersion/L in a geometric series with a separation factor of √10 by dilution with dilution water.


The test item concentrations were analytically verified via LC-MS/MS in the fresh media at the start of the exposure-renewal intervals on days 0, 7, 14, 16 (0 hours) and in the old media at the end of the exposure intervals on days 1, 8, 15, 17 (24 hours, respectively) in all concentration levels and in the control.


The measured concentrations of the test item in fresh media at the start of the respective exposure-renewal intervals (0 hours) were in the range of 78 to 98% of the nominal concentrations. At the end of the respective exposure-renewal intervals (48 and 72 hours), the measured test item concentrations in the old media were in the range of <LOQ to 29% of the nominal concentrations.


Since the measured test item concentrations were not within ± 20% of the nominal concentrations, the time weighted mean measured test item concentrations were used for the evaluation of the effect-concentrations.


The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met. To be valid, the maximum control mortality rate of parent animals must not exceed 20% and the mean offspring number of the survived females at day 21 must be at least 60.0. In the present test, the mortality was 20% and the mean offspring number of the survived females was 95.6 in the control.


The Cochran-Armitage trend-test revealed a significant trend in mortality. Thus, according to OECD 211 it is required to report the effects for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor.


 


Endpoints for Reproduction and Mortality (based on the time weighted mean measured test item concentrations and the nominal test item concentration)





























































Effect values



time weighted mean measured test item concentrations
[mg/L]



nominal test item
concentration

[mg/L]



EC10 Reproduction


(with 95% confidence limits)



0.346 (0.150 – 0.797)



0.728 (0.347 – 1.53)



EC20 Reproduction


(with 95% confidence limits)



0.460 (0.208 – 1.042)



0.973 (0.475 – 2.02)



EC50 Reproduction


(with 95% confidence limits)



0.792 (0.303 – 2.118)



1.69 (0.698 – 4.09)



LOEC Reproduction



0.984



2.08



NOEC Reproduction



0.268



0.658



LC10 Adult mortality after 21 days


(with confidence limits)



Not applicable



Not applicable



LC20 Adult mortality after 21 days


(with confidence limits)



Not applicable



Not applicable



LC50 Adult mortality after 21 days


(with confidence limits)



1.02 (0.984 - 2.41)*



2.39 (2.08 - 6.58)*



LOEC Adult mortality after 21 days



2.41



6.58



NOEC Adult mortality after 21 days



0.984



2.08



 


* The goodness of fit for this parameter is low.


 

Description of key information

21 d EC10 = 0.346 mg/L (OECD TG 211, Daphnia magna); GLP, RL1

Key value for chemical safety assessment

Fresh water invertebrates

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

Additional information

A Daphnia magna reproduction test (semi-static, 21 d) of partially unsaturated TEA-Esterquat was conducted at the test facility according to OECD 211 (2012). The test species was Daphnia magna STRAUS. Ten daphnids, held individually, were used per concentration level and control. At test start the daphnids were < 24 hours old. The study was carried out under semi-static conditions with a daily renewal. The aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.


Nominal concentrations of the test item were selected based on the results of a preliminary range finding test (non-GLP) as follows: 2.00 - 6.32 - 20.0 - 63.2 - 200 mg dispersion/L (separation factor √10), corresponding to 0.208 – 0.658 – 2.08 – 6.58 – 20.8 mg test item/L (dry content of the suspension: 10.4%) and the time weighted mean measured concentrations 0.0935 - 0.268 - 0.984 - 2.41 - 9.29 mg test item/L.


A stock solution with a nominal concentration of 200 mg dispersion/L was prepared as specified in section 5.2 and was used for the preparation of the concentration levels in the range of 2.00 - 200 mg dispersion/L in a geometric series with a separation factor of √10 by dilution with dilution water.


The test item concentrations were analytically verified via LC-MS/MS in the fresh media at the start of the exposure-renewal intervals on days 0, 7, 14, 16 (0 hours) and in the old media at the end of the exposure intervals on days 1, 8, 15, 17 (24 hours, respectively) in all concentration levels and in the control.


The measured concentrations of the test item in fresh media at the start of the respective exposure-renewal intervals (0 hours) were in the range of 78 to 98% of the nominal concentrations. At the end of the respective exposure-renewal intervals (48 and 72 hours), the measured test item concentrations in the old media were in the range of <LOQ to 29% of the nominal concentrations.


Since the measured test item concentrations were not within ± 20% of the nominal concentrations, the time weighted mean measured test item concentrations were used for the evaluation of the effect-concentrations.


The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met. To be valid, the maximum control mortality rate of parent animals must not exceed 20% and the mean offspring number of the survived females at day 21 must be at least 60.0. In the present test, the mortality was 20% and the mean offspring number of the survived females was 95.6 in the control.


The Cochran-Armitage trend-test revealed a significant trend in mortality. Thus, according to OECD 211 it is required to report the effects for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor.


 


Endpoints for Reproduction and Mortality (based on the time weighted mean measured test item concentrations and the nominal test item concentration)





























































Effect values



time weighted mean measured test item concentrations
[mg/L]



nominal test item
concentration

[mg/L]



EC10 Reproduction


(with 95% confidence limits)



0.346 (0.150 – 0.797)



0.728 (0.347 – 1.53)



EC20 Reproduction


(with 95% confidence limits)



0.460 (0.208 – 1.042)



0.973 (0.475 – 2.02)



EC50 Reproduction


(with 95% confidence limits)



0.792 (0.303 – 2.118)



1.69 (0.698 – 4.09)



LOEC Reproduction



0.984



2.08



NOEC Reproduction



0.268



0.658



LC10 Adult mortality after 21 days


(with confidence limits)



Not applicable



Not applicable



LC20 Adult mortality after 21 days


(with confidence limits)



Not applicable



Not applicable



LC50 Adult mortality after 21 days


(with confidence limits)



1.02 (0.984 - 2.41)*



2.39 (2.08 - 6.58)*



LOEC Adult mortality after 21 days



2.41



6.58



NOEC Adult mortality after 21 days



0.984



2.08



 


* The goodness of fit for this parameter is low.