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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:
03-Jun -2020 -24 Jul 2020
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Sponsor, N23062
- Purity, including information on contaminants, isomers, etc.: 100 % according to Certificate of Analysis

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability in water for at least 2 hours at room temperature protected from light is confirmed over the concentration range 5 to 50 mg/mL
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: no medium used
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: no medium used
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not applicable
Analytical monitoring:
yes
Details on sampling:
For analysis, 2.0 mL Sample from the approximate centre of the test solutions, were taken from all test concentrations and the control in followed Frequence: From freshly prepared solutions on days 0, 7, 12 and 16 and Old solutions on days 2, 9, 14 and 19.
The replicates of one renewal period were pooled at each concentration before sampling.
Additionally, reserve samples of 2.0 mL were taken from all test solutions.
Samples were stored in a freezer (≤ -15°C) until analysis.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The off-white granules of V50 were dissolved completely in test medium (M7) by vigorous shaking. Solutions (0.10, 0.32, 1.0, 3.2 and 10 mg/L) were prepared by serial dilution, starting with the highest concentration of 10 mg/L. All test solutions were clear and colorless at the end of the preparation procedure. The above procedure was performed under dimmed light conditions to minimize the exposure to light.
As control the test medium without test item or other additives were used in the Study.

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
Test species: Daphnia magna, at least third generation, obtained by a cyclical parthenogenesis
under specified breeding conditions.
Source: In-house laboratory culture with a known history (Charles River Laboratories Den Bosch
BV; Hambakenwetering 7; 5231 DD ‘s-Hertogenbosch; The Netherlands)
Age at study initiation: less than 24 hours
Feeding during test: Twice daily an amount of 0.25 mL of a Chlorella sp. On weekend days an amount of 0.50 mL was added in one single feed.This daily ration corresponded to 0.2 mg
C/Daphnia/day.

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
196-232 mg CaCO3)
Test temperature:
19-21°C
pH:
7.6-8.5
Dissolved oxygen:
7.5-9.6
Nominal and measured concentrations:
Nominal concentration: 0.10, 0.32, 1.0, 3.2 and 10 mg/L;
measured concentration as Time Weighted Mean (TWM): 0.073, 0.27, 0.81, 2.7 and 8.2 mg/L.
Details on test conditions:
TEST SYSTEM
-Test vessels (Type,Material, size, headspace, fill volume): glass vessels covered with a Perspex
plate, 60 mL (6 x Ø 3.5 cm)
- Renewal rate of test solution (frequency): Three times a week (Day 2,5,6 and 8)
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates):10
- No. of vessels per control (replicates): 20
-Volume of test medium per Vessels: minimum 50 mL

OTHER TEST CONDITIONS
- Photoperiod: Daily photoperiod of 16 h
- Light intensity: 428 -499 Lux


TEST MEDIUM
- Source/preparation of medium: ISO Medium M7 were perpared in accordance to the OECD
211 while dissolving Macro salts, salts and vitamines in laboratory tap water purified by
Reverse Osmosis. All chemicals have analytical grade.
- Culture medium different from test medium: no

WATER PARAMETERS and measuring intervals:
- Temperature, oxygen and pH At the start of the test and just before and after each renewal.
Additionally, the temperature was continuously monitored in a temperature-
control vessel.
- Hardness Once a week in fresh and old media from the control and the highest test
concentration.
- Total Organic Carbon (TOC) at the start of the test in blank test medium.


EFFECT PARAMETERS MEASURED (with observation intervals) :
- parental daphnids: number of living, immobile and dead daphnids (daily)
body Length (end of the test)

- Offspring daphnids:
Newborn daphnids: number and condition of newborn young (daily)
Appearance first brood, Presence of unhatched eggs, Incidence of
immobility, Incidence of entrapment (When observed)

RANGE FINDING STUDY:
conditions:
-10 d Range-Finding Test was performed prior to the reproduction test. One controle and three
different concentrations (0.10, 1.0, 10 mg/L) were testet with 2 replicates each and 5 daphnids
per replicate.
- recorded endpoints/observations: survivability, Number of offspring, Presence of eggs in the
brood pouch

RESULTS RANGE FINDING STUDY
- Survivability:
All daphnids exposed to the control and concentrations up to and including 1.0 mg/L survived
the ten-day test period, while a single daphnid died at the highest test concentration on day 9.
- Number of offspring:
More offspring were recorded at any test concentration when compared to the control group.
The number of offspring increased with increasing test item concentration. Hence, no adverse
effects on reproduction of Daphnia were expected based on these results.

- Presence of eggs in the brood pouch:
In all test groups, presence of eggs in the brood pouch was recorded for the first time on
day 5. The first brood appeared generally on day 8 in the control and at two lowest test
concentrations. The first brood was observed on day 7 at the highest test concentration, and
thus, appearance of the first brood was slightly earlier at this treatment when compared to the
control.
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
2.7 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.5 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
reproduction
Details on results:
In this GLP study, according to OECD guideline 211 all validity criterion are met.

MORTALITY OF PARENTAL DAPHNIDS
Three out of the twenty parental daphnids in the control group died during the test period. Not more than one parental daphnid died (i.e. ≤10% morality) at TWM concentrations up to and including 2.7 mg/L during the 21-day exposure period. No parental daphnids survived exposure to the highest concentration tested. It should be notice, that in the Range-Finding Test (10d) only one Daphnia died after exposition to the highest test Konzentration (10 mg/L), this might be due to shorter test duration.

PARENTAL GROWTH
No dose-related reduction in parental body length was found at the range of concentrations
tested. Reduction of body length did not exceed 1.9%, which was recorded at the lowest
concentration tested. There were no statistically significant differences between the control
and test concentrations.

AGE OF FIRST REPRODUCTION
The first brood was observed at day 7 in all groups. The mean age at first reproduction was
lower at all test concentrations when compared to the control, and thus, there was no need to
test for presence of a statistically significant delay in appearance of the first brood. In conclusion, the test item did not cause delay in onset of reproduction.

REPRODUCTION
The EC10 for reproduction from V 50 was 3.5 mg/L with a 95% confidence interval ranging from 1.9 to 6.2 mg/L. No toxic effect of reproduction was observed up to and including 2.7 mg/L, even though the number of offspring was higher than in control group. Only at the highest concentration tested, a statistically significant reduction of 49% was observed. On average,165 offspring were produced per introduced daphnid in the control treatment. Since none of the parental daphnids survived in this group, this reduction was attributable to lethal toxicity rather than chronic effects on reproduction.




Validity criteria fulfilled:
yes
Conclusions:
Based on the experimental observations the effect concentration for reproduction was calculated with EC10(21d) = 3.5 mg/L. All validity criteria were fulfilled and hence the study is considered adequate for regulatory purposes.
Executive summary:

In the present GLP study, according to OECD guideline 211, a EC10 (21d) for reproduction of 3.5 mg/L 2,2’-azobis[2-methylpropionamidine]dihydrochloride (V-50) was determined for Daphnia magna. The results idicated V-50 did not affect reproduction, parental growth and parental survival of Daphnia magna up to a time weighted mean concentration of 2.7 mg/L after 21 days of exposure (NOEC).
Nevertheless, Exposure to a time weighted mean concentration of 8.2 mg/L induced significant inhibition of the reproductive capacity of the parental daphnids (LOEC). It should be mentioned that this effect is attributable to complete mortality of the parental daphnids at this concentration rather than chronic effects on reproduction. The LC50 (21d) for the parental daphnids was 3.7 mg/L.

Description of key information

In conclusion, under the conditions of the present study 2,2’-azobis[2-methylpropionamidine]dihydrochloride (V-50) did not affect reproduction, parental growth and parental survival of Daphnia magna up to a time weighted mean concentration of 2.7 mg/L after 21 days of exposure (NOEC).


Exposure to a time weighted mean concentration of 8.2 mg/L induced significant inhibition of the reproductive capacity of the parental daphnids (LOEC). This effect is attributable to complete mortality of the parental daphnids at this concentration.


The EC10 for reproduction was 3.5 mg/L with a 95% confidence interval ranging from 1.9 to 6.2 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

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

Additional information

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