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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to protocol.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
no chemical analysis could be performed
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Preparation of the stock solutions:
A flow through setup was used in which the test medium flowed continually over approximately 1 g of the test substance suspended in a glass column with a glass frit under continuous agitation through which the test medium but not test chemical could pass. 1 gram of test material was replaced and the column thoroughly cleaned at least once a week to ensure a large excess of test material was always present in the column. The test substance in its un-dissolved form was known to be stable at the temperature the study was conducted. Stability data provided by the sponsor is filed in the raw data.

Preparation of the test solutions:
The test solution was automatically generated by the pumps in the flow-through system. The M4 medium was prepared in advance in a 60 L reservoir that was continually aerated. This was used to feed both the single test concentration and the control replicates via peristalstic pumps.

One peristaltic pump pumped the test medium at the desired rate from the resovoir to an overflow aquaria via a two glass columns designed to maximise water solubility. From the overflow aquaria the control medium was pumped at an equal rate to the 3 control replicates.

The test concentration was prepared in an identical fashion as detailed above except the test medium was routed via three glass columns. Column one contained a large excess of the test material (1 g). This column was continually agitated with a Teflon stirrer at high speed to maximise solubility. Liquid leaving the first column was as with the control replicate pumped over a second column with a spiral interior further mixing the test solution. Finally the test solution passed through a third column in which the solution flowed over a series of protrusions restricting the liquid flow and promoting yet further mixing. The liquid ended in an overflow aquaria from which test solution was pumped at an equal rate to the 3 control replicates. Each aquarium was calibrated at the start of the test with a flow rate of 1.4 mL/minute equal to approximately 5 aquarium turnovers per day per replicate.

All pumps were run in and then calibrated prior to the start of the study and were presumed to be stable during this period.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna clone 5 stock, (origin: Wil Research (Formerly Notox), The Netherlands). The animals used in the test were less than 24 hours old and were obtained from parent animals reproducing parthenogenetically and aged between 2-4 weeks having previously produced at least one brood before use. Offspring from the Daphnia culture were reference tested with potassium dichromate twice annually in the relevant dilution water. The culturing of the test animals was in accordance with laboratory Standard Operating Procedures.
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Test temperature:
20.65-22.05 ºC in the first test
20.75-21.15 ºC in the second test
pH:
7.7 - 8.2
Dissolved oxygen:
8.1 - 8.9 mg O2/L
Nominal and measured concentrations:
maximum water solubility
Details on test conditions:
TEST SYSTEM
- Test vessel: glass aquaria
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): One peristaltic pump pumped the test medium at the desired rate from the resovoir to an overflow aquaria via a two glass columns designed to maximise water solubility. From the overflow aquaria the control medium was pumped at an equal rate to the 3 control replicates.
The test concentration was prepared in an identical fashion as detailed above except the test medium was routed via three glass columns. Column one contained a large excess of the test material (1 g). This column was continually agitated with a Teflon stirrer at high speed to maximise solubility. Liquid leaving the first column was as with the control replicate pumped over a second column with a spiral interior further mixing the test solution. Finally the test solution passed through a third column in which the solution flowed over a series of protrusions restricting the liquid flow and promoting yet further mixing. The liquid ended in an overflow aquaria from which test solution was pumped at an equal rate to the 3 control replicates.
- Renewal rate of test solution (frequency/flow rate): flow rate of 1.4 mL/minute equal to approximately 5 aquarium turnovers per day per replicate
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

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 of ambient light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : parenet mortality, lenght and weight; reproduction, time to first brood.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: maximum water solubility
- Results used to determine the conditions for the definitive study: no effects observed at the maximum water solubility
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Remarks on result:
other: see "Any other information on results incl. table"
Details on results:
Parent animal mortality:
- First definitive test
During the first definitive test 8 animals died in the control. This is two more than is acceptable in the test guideline. 8 animals also died in the test concentration. It is clear that the test substance had no effect on parent mortality. However in order to completely meet the guideline criteria with respect to control mortality the test was repeated. Due to reproduction criteria being comfortably met and the first study being otherwise usable, data for all endpoints were still generated. The intention being, the pooling of all data for a more robust endpoint.
- Second definitive test
During the test repeat 2 animals died in the control. No mortalities were observed in the test concentration. The test repeat was conducted with juveniles from the same stock culture as the first study under identical conditions. This demonstrates that the mortality in the first study was likely due to biological variation and not due to the health of the stock culture. It is the study directors opinion that all data can be pooled for a more reliable statistical analysis.

Coefficient of variation of control fecundity:
The recommended guideline criteria for the coefficient of variation (less than 25% in the control was achieved.

Statistical results:
Although the first definitive test had slightly too much control mortality to be valid in its own right a repeat test using the same stock culture demonstrated that the culture was in good health with very low mortality in the second study. A significant amount of length, weight and reproduction data was generated in the first study which ran the full test duration. Due to both studies comfortably meeting the reproduction criteria and considering both were conducted under identical conditions all results were pooled together to generate a larger data set and give greater statistical power to the results. The test data was compared in all cases directly to the control data using a Wilcoxon Two sample test as recommended in Ref 3 when a single treatment and control are tested.

All pooled data for reproduction and dry weight showed no significant differences in comparison to the control. A significant difference in the length result was detected.

Any other biological effects observed:
The time to first brood occurred in both the control and test concentration on day 7 and on day 6 in the first and second tests respectively. The test substance is therefore considered to have had no effect on the time to first brood. No abnormalities or mortality in the neonates was observed.

It was noted that the animals in the first study were lighter than in the second study. This was both the case in the control and test replicates and is not indicative of a test substance related effect. It may however explain the elevated mortality in the first study as a result of a non optimal feeding rate or algae culture used for feeding.

In the absence of chemical analysis close attention was paid to the algae in the test system as an indirect indication of test substance presence. Sedimentation of the algae used for feeding was observed in test replicates but not in the control. In addition during the 21 days of both studies a slight growth of algae occurred in the glass tubing of the control replicate whilst the tubing in test substance replicate remained free of algae. As a result of the test substance less algae was therefore available to the organisms in the test concentration replicates.

In conclusion under flow through conditions the stability of the test substance was sufficient to influence algae growth indicating indirectly the presence of the test substance despite not having an analytical method capable of such a low level of detection. These effects were not seen in the separate algae study due to the static nature of a standard algae test and the resulting (and unavoidable) decrease in exposure during the study. These effects should not be as considered significant to algae but are worthy of note in this study as conformation that the test material was present in solution albeit at levels that were not quantifiable.
Results with reference substance (positive control):
No data
Reported statistics and error estimates:
The test data was compared in all cases directly to the control data using a Wilcoxon Two sample test.

In conclusion the lack of chemical analysis can be considered to be the greatest limitation of this study. This was due to the extremely low solubility being below the detection capabilities of the available test method. This was addressed by the use of a flow through system to ensure the best / most constant exposure possible with this test substance.

 

In total 2 (21 day) studies under GLP and one non GLP 21 day range finding study were conducted. The subsequent GLP data was pooled and analysed with a two sample statistical method. No significant differences were detected for reproduction and dry weight. No effects on time to first brood, juvenile or parent mortality was observed. Significant difference was detected for length. 

 

Considering that reproduction is considered the primary endpoint and that 5/6 of the observed endpoints show no effect the study director considers the test substance to be non toxic to Daphnia magna at its maximum achievable solubility. Furthermore considering that test animals (in test substance replicates) received less food due to interaction of the test substance with algae. Any resulting discrepancies in the secondary endpoints cannot be attributed directly to toxic effect.

 

Considering the proven ready biodegradability of the test substance the test substance is not expected to present a significant chronic aquatic hazard to invertebrates.

Validity criteria fulfilled:
yes
Conclusions:
This study is reliable without restrictions. It is performed according to OECD guideline 211 under GLP and all critical validity criteria were fulfilled.
The test substance concentration was not monitored during the test as chemical analysis was technically not feasible. The calculated endpoints are adequate for C&L and risk assessment purposes.
Executive summary:

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.

Description of key information

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 onDaphnia Magnaat its solubility limit in the test medium.

Key value for chemical safety assessment

Additional information