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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:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
REG (EC) No. 440/2008 amended by REG (EC) No. 2017/735
Deviations:
yes
Remarks:
For few individual periods, the temperature of the test media was up to 23 °C. This deviation did not adversely affect the results of this study.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Remarks:
It was not possible to implement an adequately sensitive method at the required concentration range using HPLC/ELSD and HPLC/MS with different eluents and columns, or by GC/FID (after derivatisation).
Vehicle:
yes
Remarks:
methanol
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

The preparation of the test media is based on the OECD series on testing and assessment No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

- Method: The application solution for the dosage of the highest loading rate of 3.2 mg/L was prepared by completely dissolving 32 mg of the test material in 20 mL of methanol by intense stirring for 5 minutes. This application solution was diluted with methanol to prepare the application solutions used for the dosage of the lower test concentrations.
For the preparation of the test media, 3 or 6 mL of each application solution were spiked into the empty stirring vessels. The methanol was evaporated to dryness under a nitrogen stream. Thereafter, 1500 mL or 3000 mL test water, respectively, were added into the vessels in order to disperse the test item, using ultrasonic treatment for 15 minutes and intense stirring for 24 hours at room temperature in the dark. The stirring period of 24 hours was chosen to dissolve a maximum concentration of the test item in the dispersion. After stirring, the dispersions were filtered through a membrane filter (Whatman, Type NC45, pore size 0.45 µm). The filter was pre conditioned with at least 200 mL of the respectively dispersion. The filtrates of the equilibrated dispersions were tested as WAFs. The test media were prepared just before the start of the test and prior to each test medium renewal. After filtration, the test media were clear solutions.
Additionally, the filtrate of the highest loading rate of 3.2 mg/L was tested with the addition of 10 mg/L Humic acid sodium salt (HA). For this, 8.0 mg of HA were suspended in 800 mL undiluted filtrate of the loading rate of 3.2 mg/L using further 15 min intense stirring at room temperature.

- Controls:
Blank control: test water without addition of test item, nor solvent or HA
Solvent control: 3 mL methanol without test item were spiked into the empty stirring vessel. The further procedure was the same as used for preparation of the WAFs.
HA control: 8.0 mg of humic acid sodium salt (HA) was suspended in 800 mL test water using 15 min intense stirring at room temperature.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flee
- Strain/clone: STRAUSS
- Source: in-house culture originally obtained from the Daphnia Collection of the University of Basel/Switzerland in 2015
- Feeding during test
- Food type: suspension of freshly grown green algae (Desmodesmus subspicatus) and a commercial fish diet (Tetra Min® Hauptfutter, TETRA-Werke, 49304 Melle / Germany)
- Amount: 0.20 mg total organic carbon/animal/day
- Frequency: daily

ACCLIMATION
- Acclimation period: culture medium, temperature and light conditions were identical to those in the test.
- Type and amount of food: green algae suspension (Desmodesmus subspicatus) or a mixture of this suspension and a commercial fish diet (TETRA MIN Hauptfutter, TETRA-Werke, 49304 Melle / Germany), three times a week
- Health during acclimation (any mortality observed): no mortality or signs of stress were observed and the brood stock was healthy.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Stock animals were maintained separately in a 100 mL glass beaker filled with about 80 mL culture medium and were transferred twice a week to fresh medium. The condition of the stock animals was frequently checked.
The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny. At the start of the test, the test animals were less than 24 hours old, by removing the offspring <24 hours before start of the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
2.5 mmol/L (250 mg CaCO3/L)
Test temperature:
19 - 23°C (for few individual periods, the temperature was up to 23 °C. The influence on the test can be neglected, since the time ratio of these periods was less than 5 % of the total test duration, the daily average temperature was between 19 and 22 °C and the daily variation of the temperature was within 2°C at these periods, as recommended by the guideline).
pH:
7.9 - 9.1
Dissolved oxygen:
≥ 7.7 mg O2/L
Nominal and measured concentrations:
nominal: 0.032, 0.10, 0.32, 1.0 and 3.2 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers containing 80 mL test medium, covered with glass plates
- Aeration: none
- Renewal rate of test solution: twice a week (every 48 or 72 hours)
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 80 mL/animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (Elendt M7 medium) according to the OECD Guideline No. 211. Analytical grade salts and additives were dissolved in ultrapure water.
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30-minute transition period
- Lighting: between approximately 15 and 18 µE m-2 s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- daily: immobility of adults, number of living and dead offspring and presence of aborted eggs.
- test end: body length of surviving adults (measuring the daphnids from the top of the head to the basis of the spina with the use of a binocular microscope).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Results used to determine the conditions for the definitive study:
14-day range finding study at nominal loading rates of 0.04, 0.20, 1.0 and 5.0 mg/L.
- adult mortality: 100% after 48 hours at 5.0 mg/L;
- inhibition of reproduction: 13%, 19% and -4% at 0.04, 0.20 and 1.0 mg/L
Reference substance (positive control):
not required
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: results are expressed in terms of loading rate
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
0.84 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% confidence limits: 0.33-1.2 mg/L; results are expressed in terms of loading rate
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks on result:
other: results are expressed in terms of loading rate
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
> 3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks on result:
other: 95% confidence limits: could not be determined; results are expressed in terms of loading rate
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidenceresults are expressed in terms of loading rate
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
0.53 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence limits: 0.09 and 0.94 mg/L; results are expressed in terms of loading rate
Details on results:
- Behavioural abnormalities: none
- Observations on body length: no detrimental effect on the body length was observed. The mean body length of the daphnids in the controls and in the treatments up to and including the loading rate of 1.0 mg/L was in the range of 4.93-5.0 mm (corresponding to 99-100% compared to the blank control). At the highest loading of 3.2 mg/L, the mean body length was 4.53 and 4.75 mm (91% and 95%) in the absence and presence of humic acid, respectively.
- Other biological observations: Mortality of adult daphnids: No biological relevant mortality (0-10%) was observed at up to and including the loading rate of 1.0 mg/L and at the loading rate of 3.2 mg/L containing humic acid. At the loading rate of 3.2 mg/L without humic acid, adult mortality was 80%.
- Mortality of control: No biological relevant mortality (0-10%) was observed in the blank control, the solvent control and the humic acid control
- Other adverse effects control: none
- Abnormal responses: none

The detailed biological results obtained for reproduction and parental mortality and body length are presented in "Attached background material" - "Tables & Figures_long-term toxicity to daphnia AS-400112 (IES 20170067).pdf".



Reported statistics and error estimates:
Statistical analysis was performed using ToxRat Professional®, Version 3.2.1 (ToxRat® Solutions GmbH).

According to OECD Guideline No. 211 (2012), if mortality was lower or equal 10% the reproduction rate was calculated as the total number of living offspring produced per surviving parent female. For higher (test substance dependent) mortality the reproduction rate was calculated as the total number of living offspring produced per parent female introduced in the test.

Comparison of the mean reproduction rate and the body length of the daphnids in the solvent control and in the HA control with the blank control by a Student-t test ('a' = 0.05, two-sided) showed no significant differences. Thus, statistical evaluations were performed using the blank control.

The mean reproduction rates and the mean body lengths of the daphnids at the loading rates were compared with the blank control by multiple Welch t-tests and Williams t-test, respectively (each with 'a'= 0.05, one-sided).

The NOEL for mortality was determined directly from the raw data.

Additionally, the EL values for inhibition of reproduction rate and mortality after 21 days were calculated by Probit Analysis using linear maximum likelihood regression based on the mean values per loading rate level.

The EL10 and EL50 of the body length after 21 days could not be calculated due to the low toxic effect of the test item on the body length up to the highest loading rate.
Validity criteria fulfilled:
yes
Remarks:
validity criteria for the controls: mortality of the parent animals was 0-10% at the end of the test (≤20%), mean surviving offspring per parent animal was 162-174 (≥60) and the coefficient of variation was 4.9-7.6% (<25%)
Executive summary:

The impact of the UVCB substance AS-400152 (synonym WS400152) on the survival, reproduction rate and growth (body length) of Daphnia magna was investigated in a semi-static test over 21 days following the OECD Guidelines for Testing of Chemicals, No. 211 (2012) and EU C.20 (2017). D. magna were exposed to five concentrations of AS-400152 ranging from 0.032 – 3.2 mg/L prepared as water accommodated fractions (WAFs). For preparation of the WAFs, the respective amounts of test material were added from stock solutions in methanol (the solvent was evaporated prior to addition of test water). To mimic environmental conditions, the influence of the test material at the loading rate of 3.2 mg/L was additionally tested in the presence of humic acid sodium salt (HA). In parallel, a blank, a solvent and a HA control were run.

 

No differences for survival, reproduction rate and body length were observed between the blank, solvent and humic acid control. Thus, statistical evaluations were performed using the blank control.

 

Reproduction rate was significantly reduced at 1.0 mg/L, growth and survival at 3.2 mg/L (the highest loading rate). At 3.2 mg/L, only two out of ten parents survived.

 

In the presence of HA, all test animals survived at the highest loading rate (3.2 mg/L), whereas reproduction rate and body length were statistically reduced. However, the presence of HA clearly reduced the toxic effects of the test material on the reproduction rate from 71% to 21%. Beyond that, the two surviving Daphnia did not differ from the control animals with regard to the mean reproduction rate (167 living offspring and 174, respectively

 

In conclusion, taking into account the effects of AS-400152 on survival, growth and reproduction, the overall 21-day NOEL was the loading rate of 0.32 mg/L. The presence of humic acid sodium salt partly mitigated the toxic effect on Daphnia magna.

Description of key information

Overall 21-d NOEL (based on loading rates) 0.32 mg/L for Daphnia magna (OECD 211, EU C.20)

Key value for chemical safety assessment

Additional information

NOEL for freshwater invertebrates: 0.32 mg/L