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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:
November 2019 - February 2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Established CROs working under GLP conditions were not able to set-up the very complicated substance analytics and measure the adsorbed substance in the daphnia feed (green algae). This special set-up was only possible in an in-house laboratory of one of the co-registrants of the substance (Nouryon). This laboratory does not have a certification under GLP for this type of studies, but works under an equivalent quality control system.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
GLP compliance:
no
Remarks:
The test was carried out in compliance with the OECD principles of Good Laboratory Practice.
Analytical monitoring:
yes
Details on sampling:
- Instead of water phase only analytical measurements, the water and the test material adhering to the algae were quantified for a better quantification of the test material in the test system. For this reason, hydrolysis products were not quantified due to sufficient quantifiable parent being present.

SAMPLING
Clean liquid samples were sampled with a pipette and diluted 50:50 with THF to prevent adsorption to the glass sample vials. Further dilution was conducted in THF if required. 20 mL samples containing algae were filtered with a 0.45 μm Whatman PTFE filter. The filtered algae was then extracted with 10 mL of THF and then analysed identically to the water samples, correcting for any concentration or dilution steps. The filtrate (test medium passing through the filter) was analysed as a water sample to allow an estimation of the non-bound fraction of the test material.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A new stock solution of approximately 2 g/L was made of the test material weekly in acetone. This was dosed to the 500 mL or 1 L of test medium in a sufficient quantity so as to achieve the desired test concentration of 120 μg/L. A sufficient amount of test solution was made to fill all of the beakers and analytical parallels as required. The required amount of algae was added to the test medium and was thoroughly stirred to ensure a homogeneous distribution of the algae. The test medium was then added to the test vessels. The same procedure was repeated for every solution refreshment. The controls were prepared in the same manner but without the test substance. A larger volume was prepared for the weekends.

- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium: 100 µL/L
- Other relevant information:
The test substance was known to have low toxicity and adsorb strongly. In order to maximize the chance of achieving a measured exposure a test concentration of 120 μg/L was chosen for testing, which is higher than that would be expected based on the expected water solubility (5 μg/L). Due to the properties of the test material the test material binds strongly to organic material and exposure occurs primarily via a feeding route. However, despite binding the test material does remain in the test system and exposure does occur via a relevant exposure route. The concentration of the bound fraction was therefore determined in fresh and old solutions. This can be considered as a worst-case approach for the test substance far in excess of concentrations achievable if dosed at its solubility limit. Standard and acetone controls were also prepared.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: STRAUS Clone 5
- Age at study initiation: less than 24 hours old
- Source: Charles River Laboratories, The Netherlands
- Age of parental stock: 2 - 4 weeks
- Feeding during test: yes
- Food type: algae species Chlorella vulgaris
- Amount: 0.1 mg/C per 50 mL for the first 2 days and the increased to 0.2 mg/C per 50 mL per day
- Frequency: daily

ACCLIMATION
- Acclimation period: not needed, culture is held at the same conditions as the test
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Test temperature:
20.3 - 21.0 °C
pH:
7.2 - 8.4
Dissolved oxygen:
7.1 - 9.4 mg O2/L
Conductivity:
<10 μS/cm
Nominal and measured concentrations:
Nominal concentration: 120 µg/L
Measured concentration: 44.7 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: beakers
- Type: covered with a glass plate to minimize evaporation
- Material, size, headspace, fill volume: glass, 50 mL on weekdays and 100 mL on weekends to provide sufficient food for the weekend
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): 3 times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 medium according to OECD 211
- Non-purchable organic carbon (NPOC) content: <0.2 mg/L
- Metals: Copper: <10 μg/L
- Conductivity: <10 μS/cm
- Culture medium different from test medium: no, the same

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 10 - 20 μmol m-2 S-1

EFFECT PARAMETERS MEASURED:
- reproductive capacity expressed as the total number of neonates per adult daphnid at the end of the test
- length and dry weight
- parent and juvenile mortality
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
44.7 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
44.7 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
length
Details on results:
- Parent animal mortality in the test concentration: No parent animals died at the only test concentration. No dose response mortality occurred.
- Behavioural abnormalities: none
- Other biological observations: none
- Mortality of control: no parent died in the control, one parent Daphnia dies in the solvent control
- Other adverse effects control: none
- Immobilisation of control: none
- Abnormal responses: none
- Any observations that might cause a difference between measured and nominal values: adsorption of the test item on the algae biomass
Statistical evaluation of the reproduction data and length and weight of parent animals at end of the study

Reproduction (Primary endpoint)
No statistical difference was detected between the solvent control and the only test concentration. The NOEC for reproduction can therefore be expressed as 120 μg/L (nominal) or as 44.7 μg/L (estimated time weighted mean) concentration. Due to these values exceeding the water solubility of the test substance the test material may also be concluded to have no effects on the reproduction of Daphnia magna at its water solubility limit. ECx values were not possible to calculate due to their being no dose response in the data set.



Length
No statistical difference was detected between the solvent control and the only test concentration. The NOEC for reproduction can therefore be expressed as 120 μg/L (nominal) 44.7 μg /L (estimated time weighted mean) concentration. Due to these values exceeding the water solubility of the test substance, the test material may also be concluded to have no effects on the length of Daphnia magna at its water solubility limit. ECx values were not possible to calculate due to their being no dose response in the data set.


 


Dry Weight
Sufficient replicates for a valid statistical analysis were not available. However, the average dry weight per daphnia at the only test concentration exceeded that of the control. Supporting the findings of the other two endpoints already described.


 

Chemical analysis

The analytical method was found to be sufficiently sensitive and robust for determining the test substance concentration in this study at t = 0, 48 and 72 hours between refreshment making a time weighted mean calculation possible. However, the instable nature of the test material and its poor water solubility and adsorbing properties do mean that analysis of nominal concentrations lower than 120 μg/L will result in greater variability and difficulty in analysis. However, if a sufficiently large sample is taken from the test replicate (20 mL) detection of nominal concentrations reproducibly down to 10 μg/L is considered feasible. The chemical analysis demonstrated a good level of initial nominal recovery and complete absorption to the algae biomass. No test material in the water phase could be detected.


 


Table 1.The number of living juveniles per parent animal at each nominal test concentration, the mean number of neonates per concentration and the coefficient of variance.




















































































Reproduction Day 21



Daphnia No.



Control



120 µg/L



1



231



226



2



231



242



3



226



174



4



239



162



5



188



181



6



209



240



7



221



166



8



203



159



9



211



177



10



M



247



Total



1959



1974



Mean



218



197



Stdev



16



37



CV%



7



19



M = Mortality parent animal


 


Table 2: Length of the test organisms in standard units (Day 21)


























































 Control



120 µg/L



3.1



3.1



3.1



3.2



3.1



3.1



3.2



2.9



3



3



3



3.2



3.1



3



2.9



3



3



3.2



3



3



Mean



Mean



3.05



3.07



The measurement took place via a standard scale in the binocular microscope eyepiece. This measurement is unitless and changes dependent on magnification used. It is used for comparison purposes only to determine if a length difference occurs.


 


Table 3: The dry weight of the test organisms (Day 21)































































Conc. µg/L



Replicate



M (dish full) (g)



M dish empty (g)



No. of daphnids



Weight  (mg)



Average weight (mg)



Control



I



0.70422



0.7025



5



0.344



0.33


 



II



0.70126



0.6997



5



0.312



Acetone



I



0.6908



0.6892



5



0.32



0.31


 



II



0.69537



0.6942



4



0.2925



120



I



0.69721



0.695



5



0.442



0.452


 



II



0.69941



0.6971



5



0.462



 


Chemical Analysis Results










































































































Sample



Concentration of the test item (in µg/L)



T = 0



T = 24 h



T = 48 h



T = 72 h



control, water phase



< LOQ



n.d.



n.d.



n.d.



control, algae extract



< LOQ



n.d.



n.d.



n.d.



control M4, water phase



< LOQ



n.d.



n.d.



n.d.



Demi water spiked rep. 1



128.4



n.d.



n.d.



n.d.



Demi water spiked rep. 2



112.4



n.d.



n.d.



n.d.



Demi water spiked rep. 3



113.3



n.d.



n.d.



n.d.



120 µg/L rep. 1 water phase



< LOQ



n.d.



n.d.



n.d.



120 µg/L rep. 2 water phase



< LOQ



n.d.



n.d.



n.d.



120 µg/L rep. 3 water phase



< LOQ



n.d.



n.d.



n.d.



120 µg/L rep. 1 algae extract



94.3



43.2



27.7



11.0



120 µg/L rep. 2 algae extract



101.1



44.6



24.6



19.6



120 µg/L rep. 3 algae extract



92.7



51.5



25.6



14.6



n.d. = not determined


    

 


Analytical method validity


The analytical method was shown to be capable of detecting >76 to <120 % of the nominal concentration of the test material in both from the M4 daphnia test medium containing Chlorella vulgaris and in deionized water. Triplicate measurements showed an acceptable level of variation both from the water phase determination and when the test material is extracted from the filtered algae material.  


 


Time weighted average estimation





































































































Days



T = 0 h



48 h



 Geom. mean



72 h



Geom. mean



Estimated TWA


 

94.3



27.7



51.1088



11



32.2071


 
 

101.1



24.6



49.8704



19.6



44.5147


 
 

92.7



25.6



48.7147



14.6



36.7889


 
       

Average 12 d (57 % of test)



49.8980


  

28.4418


       

Average 9 d (43 % of test)


  

37.8369



16.2699


       
       
     

TWA



44.7117



 

Validity criteria fulfilled:
yes
Conclusions:
Based on a worst-case exposure far in excess of a realistically achievable environmental concentration, no effects to any of the assessed endpoints were observed, therefore, the NOEC was determined to be 120 µg/L (nominal) or 44.7 µg/L (TWA).
Executive summary:

In the course of this study the toxicity of the test substance dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static limit test according to OECD Guideline No. 211 (OECD, 2012), was assessed.
The primary test criterion used to indicate the toxicity of the test substance was reproductive capacity expressed as the total number of neonates per surviving parent animal at the end of the study. The test was conducted at a single limit test concentration of 120 µg/L. A test replicate using test medium and 100 μL/L of acetone served as the control as well as an additional control in test medium only. Data on parental length and dry weight was also generated for use in determining a secondary endpoint as additional information.
No statistical difference was detected between the reproduction in the solvent control and the only test concentration. The NOEC for reproduction can therefore be expressed as 120 μg/L (nominal) or as 44.7 μg/L (estimated time weighted average) concentration.
No statistical difference was detected between the length of the adult organisms in the solvent control and the only test concentration. The NOEC for length can therefore be expressed as 120 μg/L (nominal) or as 44.7 μg /L (estimated time weighted average) concentration.
The quality criteria for the performance of the test met the guideline requirements and the analytical method used was able to detect the test material with sufficient sensitivity, accuracy and reproducibility.
The test material was concluded to have no chronic effects on Daphnia magna up to its solubility limit in test medium and was concluded to partition completely to the algae biomass and was not detectable in the water phase but was detectable after extraction from the algae biomass.

Description of key information


Based on a worst-case exposure far in excess of a realistically achievable environmental concentration, no effects to any of the assessed endpoints were observed, therefore, the NOEC was determined to be 120 µg/L (nominal) or 44.7 µg/L (TWA).


Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
NOEC
Effect concentration:
44.7 µg/L

Additional information


In the course of this study the toxicity of the test substance dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static limit test according to OECD Guideline No. 211 (OECD, 2012), was assessed.
The primary test criterion used to indicate the toxicity of the test substance was reproductive capacity expressed as the total number of neonates per surviving parent animal at the end of the study. The test was conducted at a single limit test concentration of 120 µg/L. A test replicate using test medium and 100 μL/L of acetone served as the control as well as an additional control in test medium only. Data on parental length and dry weight was also generated for use in determining a secondary endpoint as additional information.
No statistical difference was detected between the reproduction in the solvent control and the only test concentration. The NOEC for reproduction can therefore be expressed as 120 μg/L (nominal) or as 44.7 μg/L (estimated time weighted average) concentration.
No statistical difference was detected between the length of the adult organisms in the solvent control and the only test concentration. The NOEC for length can therefore be expressed as 120 μg/L (nominal) or as 44.7 μg /L (estimated time weighted average) concentration.
The quality criteria for the performance of the test met the guideline requirements and the analytical method used was able to detect the test material with sufficient sensitivity, accuracy and reproducibility.
The test material was concluded to have no chronic effects on Daphnia magna up to its solubility limit in test medium and was concluded to partition completely to the algae biomass and was not detectable in the water phase but was detectable after extraction from the algae biomass.


 



Supporting data from a read-across substance are additionally provided:


In order to assess the toxicity of Dihexadecyl peroxodicarbonate in an aquatic environment, a Daphnia magna reproduction test under flow through conditions at the maximum achievable solubility in test medium was conducted in accordance with OECD Guideline No. 211. Testing was conducted in compliance with OECD principles of Good Laboratory Practice. Two modifications to the guideline were applied. Due to the lack of toxicity in acute and chronic preliminary testing, a single concentration at the maximum achievable concentration of the test substance in the test medium was tested. Due to the extremely low solubility of the test substance, chemical analysis was not possible. A flow through system was therefore used to ensure continual exposure and the testing of a realistic worst case scenario. The primary test criterion of toxicity used was reproductive capacity, expressed as the total number of neonates per replicate the end of the test. Secondary endpoints based on time to first brood, juvenile and parent mortality, dry weight and length were also compared in the same manner directly to the corresponding control data for significant differences.


The following validity criteria were respected:


- The average number of juveniles per surviving parent in the control was >60 in both definitive tests.


- No ephippia or abnormal mortality or presence of male daphnia occurred in the culture or in the control for both definitive tests.


- The coefficient of variation in the controls were all < 25 %.


- The mortality in the control did not exceed 20% in the second definitive test.


 


The following validity criterion was not met:


- In the first definitive test the control mortality was exceeded slightly.


 


Due to the slightly elevated mortality in the control the study was repeated in the form of a second identical definitive test. Generated test data was then pooled due to both tests being having run for the full 21 day period with good control reproduction.


No significant differences from the control were detected for reproduction and dry weight. No difference in time to first brood was observed and no juvenile or parent mortality was observed. The data for length did show significant difference from the control. Due to the vast majority of the data indicating no effects, the test substance was concluded as having no chronic effects on Daphnia Magna at its solubility limit in the test medium.

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