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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
No analytical monitoring. 50% additional iron was added prior to using the WSF. The instability of the vitamin was addressed by adding vitamins to the medium just prior to use. Statistical analysis was conducted after 20 days not 21 days.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1012011395
- Expiration date of the lot/batch: 04/01/2021
- Appearance: White soft paste
- Purity: 55 %
Analytical monitoring:
no
Remarks:
technically not feasible due to the low water solubility of the test substance
Details on sampling:
N.A.
Vehicle:
no
Details on test solutions:
Preparation of the stock solutions:
Due to inherent properties of the test material, preparation of stock solutions in the standard manner was not carried out.

Preparation of the test solutions:
Between 0.1000- 0.1002 g of the test material was loaded to 10 L test medium to generate the desired dissolved fraction of the test material. The solutions were left to stir slowly for 48 hours at room temperature in the dark prior to use. Each 10 litre vessel was used for up to a maximum of 6 solution replenishments with a new WSF being made weekly for the subsequent weeks refreshments in the same manner. In order to minimise the change in the composition of the solutions, vessels were refrigerated after use until a few hours before the next solution change. Solutions were then removed from the refrigerator and allowed to equilibrate to test temperature prior to each use whilst stirring. During the weekend periods the WSF’s stood for a longer time at room temperature and were not refrigerated.

After stirring was stopped the test solutions were left for 1-2 hours to rest before dispenser pipettes with the pipe positioned in the centre of the vessel were used for transfer of the dissolved fraction of the test solution only. No floating or sunken material was transferred. The algae, vitamin and additional iron components of the test medium were added while the test solutions were aerated. Alternatively if the test vessels had been replaced algae could also be added directly to test vessels. The solutions were renewed in this manner 6 times a week during the test. 3 times a week the test vessels were renewed together with the test solutions. 3 times a week as much of the old solutions as possible was removed with a pipette and topped up with new test solution. This was done to avoid the discarding of uneaten algae and hence the inadvertent reducing of the feeding rate.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna clone 5 stock, (Origin: WIL Research Europe, The Netherlands) cultured in conformity with the relevant SOP. The parent animals were cultured in test medium. The animals used in the test were less than 24 hours old and were obtained from parent animals reproducing parthenogenically and having an age of 2-4 weeks (having previously produced at least one brood before use). The culture is checked half-yearly for sensitivity by a reference test with potassium dichromate and is only used when guideline criteria are met.
Culture and test animals were fed a diet of 0.2 mg of carbon per daphnid 6 days per week, in the form of a suspension of the algal strain Chlorella vulgaris. The strain is cultured in the Environmental Chemistry laboratory and total organic carbon content had previously been measured.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Remarks on exposure duration:
Although the test duration was 21 days evaluation took place on the 20 day data omitting the last brood due so as to avoid incomplete laying of the last brood influencing the data. Parental length was measured on day 21.
Hardness:
12.7 °dH.
Test temperature:
20.4 - 21.5 °C
pH:
7.3 - 8.2
Dissolved oxygen:
8.2 - 9.0 mg O2/L
Nominal and measured concentrations:
10 mg/L loading rate
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers
- Type: open
- Aeration: no
- Renewal rate of test solution: daily, six times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 20
- No. of vessels per control (replicates): 20

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light
- Light intensity: 14.3 µmol/s/m2

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : number of offspring, parent mortality, parental length
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
20 d
Dose descriptor:
NOELR
Effect conc.:
< 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
20 d
Dose descriptor:
EL10
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: parental length
Details on results:
Parent animal mortality:
Two animals died in the control and no animals died at the only test substance concentration.

Reproduction:
For the primary endpoint reproduction significant difference from the control (5.5% reduction in reproduction) was detected using the students T test. The surviving adults only were used for endpoint derivation as no test substance related mortality of the parent animals was observed.

Parental length:
The students T test detects no statistical difference in the length of the test organisms.

The data is therefore somewhat contradictory. However it may be expressed as NOELR < 10 mg/L and EL10 is > 10 mg/L. Furthermore a clear effect of the test substance is usually also supported by the secondary endpoint that was not observed in this case. See conclusion for further discussion.

Any other biological effects observed:
A single animal in the control was observed as visibly smaller than the others test animals. No other biological effects were observed.
Reported statistics and error estimates:
The data on reproduction and parental length were tested for statistical significance using ToxRat professional. Version 2.10.
Validity criteria fulfilled:
yes
Conclusions:
The required test validity criteria were met. Generated data is however contradictory. A slight (5.5%) but significant effect was detected during this study. Normal practice would be to proceed to a test with a complete concentration range. However due to the substance being extremely poorly soluble this is not possible as the highest achievable concentration of the test substance has in theory already been tested. Interpretation is further complicated by the lack of chemical analysis.

In order to investigate this in more detail and conclude the best possible next steps for this test material, (non GLP) data was generated on an analogue of this test substance (CAS 94-36-0) Di benzoyl peroxide due to its better solubility. The rapid hydrolysis of CAS 94-36-0 is already documented and one could conclude similar behavior for the test substance in this study. Study NG16023 WS demonstrates that the maximum solubility of CAS 94-36-0 was achieved in demineralized water within 48 hours using a slow stir method. However using the same method CAS 94-36-0 could not be determined in M4 daphnia test medium. Indicating extremely rapid degradation in M4 medium. It is fair to conclude that during this study primarily degradation products generated from 10 mg/L loading of the test material were likely to have been tested and little or no parent material. This does not invalidate the study due to the WSF method testing the influence of water soluble degradation products. However due to the lack of analysis the data generated does therefore have limited use.

Due to the rapid hydrolysis and instability in test medium there is a high likelihood of rapid degradation in an environmental situation. The more stable and soluble degradation products of the test substance are therefore of greater relevance in determining the chronic aquatic hazard. A follow up study or literature data on degradation product (most likely 2,4,- di-chloro benzoic acid) would be required in order to definitively conclude the chronic aquatic hazard of this substance to Daphnia magna via its degradation products combined with the data from this study.

Considering the fact that this study was conducted at loading rates much higher than the classifiable chronic toxicity cutoff of 1 mg/L and still only produced a borderline result, the study director concludes that the test substance need not be classified as chronically toxic to Daphnia magna at this time. A prompt follow up with appropriate supporting data on the degradation product is recommended to allow a definitive conclusion to be made.
Executive summary:

The purpose of this study was to assess the toxicity of the test substance dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static test complying with the OECD Guideline No. 211 (OECD, 2012). The total test period was 21 days. The test solutions were renewed daily throughout the test on weekdays and once during the weekend.

 

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. Data on parentallength was also generated.

  

The nominal concentrations used in the study were as follows: 10mg/L and a control.

All concentrations given refer to the technical product as supplied by the sponsor.

 

Chemical analysis could not be carried out due to the extremely low solubility (<2µg/L) of the test material being below the detection limit of the available analytical method.

 

The following quality criteria were met:

·        Cultures were in good health (i.e. disease free, no ephippia or males, no discolored animals, valid reference test).

·        Twoparent animals died in the control group during the test period, which is not more than 20%.

·        The average number of living neonates per parent animal alive at the end of the test in the control was 171 after 20 days (minimum acceptable = 60).

·        The coefficient of variation of the average number of living neonates produced per parent animal alive at the end of the test in the control group did not exceed 25%.

 

The study generated contradictory effect data and requires confirmation by further testing. Due to a significant effect being observed (5.5% reproduction inhibition) the chronic toxicity of the test substance can be expressed as NOELR < 10 mg/L and EL10is > 10 mg/L. A clear effect of the test substance is usually also supported by the secondary length endpoint. No significant difference in parent length was observed in this case. See conclusion for further discussion and recommended follow up testing.

 

Due to the worst case approach of the testing (tested 10 x above the 1 mg/L chronic classification cutoff) and based on the data currently available, the substance should not be classified as toxic to Daphnia magna.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
The study does not claim GLP compliance. Raw data was quality checked by experienced GLP trained technicians involved in the study. While chemical analysis was not conducted under GLP it was conducted in a GLP accredited facility.
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sigma-Aldrich, S34634V
- Purity:98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal test concentrations: 0.31, 1.0, 3.2, 10.2, 32.7 mg/L including a test medium only control.
- Sampling method: Samples of all new and old test concentrations and stock solutions were taken during the test.
- Sample storage conditions before analysis:
Vehicle:
no
Details on test solutions:
Preparation of the stock solutions
The test material has a reported solubility of approximately 360 mg/L. 2 x stock solutions at 300 mg/L
were initially trialled for the first few stocks solution without complete success. Very small amounts of
test material remained visible. After which the stock solution was lowered to 200 mg/L for which a more
homogeneous stock was achieved. All subsequent stocks were prepared at 200 mg/L. Stock solutions
were stirred thoroughly ultrasonicated twice at low-moderate power for 30 seconds – 1 min. The stock
was kept turning rapidly during use.

Preparation of the test solutions
Test solutions were prepared by further dilution of the relevant stock solutions with test medium in
volumetric flasks. All pipetting took place while stock was under agitation and all pipettes were rinsed at
least once with the stock and the contents discarded. Test vessels were filled directly from volumetric
flasks immediately after preparation. The solutions were renewed three times a week during the test
each time with freshly made stock solutions using the same method as previously described.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna clone 5 stock, (Origin: WIL Research Europe, The
Netherlands) cultured in conformity with the relevant SOP. The parent animals were cultured in test
medium from the day they were born.
The animals used in the test were less than 24 hours old and were obtained from parent animals
reproducing parthenogenically and having an age of 2-4 weeks (having previously produced at least
one brood before use). The culture is checked half-yearly for sensitivity by a reference test with an
appropriate reference toxicant. The most recent reference test results were within the range given in the
test guideline. Furthermore there was no abnormal mortality or winter egg formation. The culture was
therefore deemed suitable for use.

Feeding during test:
Test animals were fed a diet of 0.1 to 0.2 mg carbon per daphnid per week day (in 50 mL) or 0.2 to 0.4
mg (in 100 mL) (Friday to Sunday) during weekends, in the form of the algal strain Chlorella vulgaris.
The strain is cultured in the Environmental Chemistry laboratory and total organic carbon content has
previously been measured.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Test temperature:
21.1 - 22.3 C
pH:
7.4 - 8.4
Dissolved oxygen:
8.7 -10.9 mg O2/L
Nominal and measured concentrations:
Nominal test concentrations: 0.31, 1.0, 3.2, 10.2, 32.7 mg/L

The concentrations measured in the stock solutions throughout the test varied from 71-95% (not
storage corrected) of the nominal concentrations. The stability samples at 10.2 mg/L indicated that only
40% of the nominal value was measured after storage. Measurements of freshly prepared solutions
demonstrated that if measured straight after preparation recovery of close to 100% if the nominal
concentrations could be recovered. The lower recoveries in the stored samples are therefore concluded
to be due to instability during the relatively long storage period.
Due to there not being any detectable biological effects in the chosen concentration range only the
highest concentration was analysed in order to represent a worst case NOEC. For the purpose of
getting a corrected average measured exposure at the highest test concentration the recovery in the
stability samples at 10.2 mg/L have been used for correction purposes. The corrected recovery of 118%
is considered a slight overestimation but falls within the acceptable range of 80-120% for which use of
nominal concentrations is accepted according to the guideline.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers
- Type: covered by glass plates during the test
- Material, size, headspace, fill volume: glass, 50ml filled with ~ 50 ml
- Renewal rate of test solution (frequency/flow rate): The solutions were renewed three times a week during the test
each time with freshly made stock solutions using the same method as previously described.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates):10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 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 due to there being no test substance related parent mortality. Data on parental length and dry-weight was also generated for use in determining secondary endpoints as required. Sufficient replicates were not present to analyse the dry weight data and average only was therefore calculated for comparative purposes only.
Reference substance (positive control):
not specified
Remarks:
The culture is checked half-yearly for sensitivity by a reference test with an appropriate reference toxicant. The most recent reference test results were within the range given in the test guideline.
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 32.7 mg/L
Nominal / measured:
meas. (not specified)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 32.7 mg/L
Nominal / measured:
meas. (not specified)
Basis for effect:
growth
Details on results:
Parent and animal mortality:
Two parent animals died in the control and at 10.2 mg/L, one died at 0.3 and 3.2 mg/L, four died at 1.0
mg/L and no mortality was observed in the highest concentration of 32.7 mg/L.

Reproduction (Primary endpoint)
The EC10 and EC50 for reproduction were not determined due to there being no statistically significant
effects at the highest test concentration. The NOEC for reproduction can therefore be expressed as
32.7 mg/L based on measured concentrations.

Length
The EC10 and EC50 for length were not determined due to there being no statistically significant effects
at the highest test concentration. The NOEC for length can therefore be expressed as 32.7 mg/L based
on measured concentrations.

Weight
Sufficient replicates for a valid statistical analysis could not be generated by this data. When reviewing
the data manually without the use of statistical software a lack of effects at the highest concentration is
clearly visible. The author concludes that the NOEC for dry weight can therefore also be expressed as
32.7 mg/L based on measured concentrations.
Reported statistics and error estimates:
The length, reproduction and mortality data were inputted into Toxrat V2.10 statistical using the OECD 211 template and the complete reports were added to the raw data. For the reproduction endpoint juveniles from living adults were calculated as no dose response effect was observed for adult mortality.
Validity criteria fulfilled:
yes
Conclusions:
The study met the biological and analytical validity criteria for a valid test and is expected to be a valid representation of the effects of the test material to the test organism in fresh water. Chemical analysis allowed the fresh and old solutions to be quantified and exposure results were expressed as nominalconcentrations due to a good recovery and stable exposure to the test substance between
refreshments. The tests substance should be considered of very low toxicity to Daphnia magna with a NOEC ≥ 32.7
mg/L. In conjunction with the data in the existing GLP study on bis (2,4-dichlorobenzoyl) peroxide (Kean, 2016) it can be concluded that the main hydrolysis product of (2,4-dichlorobenzoyl) peroxide should not be classified for chronic toxicity to Daphnia magna.
Executive summary:

The purpose of this study was to assess the toxicity of the test substance dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static test complying with the OECD Guideline No. 211 (OECD, 2012). 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 due to there being no test substance related parent mortality. Data on parental length and dry-weight was also generated for use in determining secondary endpoints as required.

The nominal concentrations used in the study were as follows:

0.31, 1.0, 3.2, 10.2, 32.7 mg/L including a test medium only control.

All concentrations given refer to the technical product as supplied by the sponsor.

The following quality criteria were respected:

- Cultures were in good health (i.e. disease free, no ephippia or males, no discolored animals, valid reference test).

- Two parent animals died in the control group over the test period, which is not more than 20%.

- The average number of juveniles per parent animal alive at the end of the test in the control was 162 after 21 days (minimum acceptable = 60)

- Analytical quality criteria for a robust method were met

- The validity criterion for the coefficient of variation (less than 25% in the control based on the number of living neonates for each parent animal alive at the end of the test) was achieved.

The measured mean exposure concentrations (after correction for storage) were between 80 and 120% of the nominal values. Nominal concentrations were therefore used for expression of the toxicological endpoints.

Description of key information

The study generated contradictory effect data. Due to a significant effect being observed (5.5% reproduction inhibition) the chronic toxicity of the test substance can be expressed as NOELR < 10 mg/L and EL10is > 10 mg/L. A clear effect of the test substance is usually also supported by the secondary length endpoint. No significant difference in parent length was observed in this case.  

A chronic Daphnia study according to guideline (OECD 211) is available. Based on the data currently available, the substance should not be classified as toxic to Daphnia magna. The EL10 > 10 mg/L.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
10 mg/L

Additional information

One OECD 211 GLP study is available.

In order to predict the effects of Bis (2,4-dichlorobenzoyl) peroxide in an aquatic environment, a long-term toxicity test to Daphnia magna was conducted in accordance with OECD test guidelines and with the OECD Principles of Good Laboratory Practice. Some modifications to the guideline were applied due to inherent properties of the test chemical.

The toxicity of the test chemical to Daphnia Magna prepared as a Water Accommodated Fraction (WAF) was determined in a semi static system over an exposure period of 21 days at a loading concentration of 10 mg/L.

The study generated contradictory effect data. Due to a significant effect being observed (5.5% reproduction inhibition) the chronic toxicity of the test substance can be expressed as NOELR < 10 mg/L and EL10is > 10 mg/L. A clear effect of the test substance is usually also supported by the secondary length endpoint. No significant difference in parent length was observed in this case.  

Due to the worst case approach of the testing (tested 10 x above the 1 mg/L chronic classification cutoff) and based on the data currently available, the substance should not be classified as toxic to Daphnia magna.