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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:
From 2019-08-08 to 2019-10-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: 707001
Purity: 98.5%
Analytical monitoring:
yes
Details on sampling:
During the test, the concentrations of the test chemical were analyzed once a week in fresh and old media(i.e. analyses were made on a sample from the same solution when freshly prepared and at renewal). 0, 9, 14 d of fresh media and 2, 12, 16 d of old media were taken from test groups and control group to analysis. For sampling of the old test media, one replicate of the ten replicates was sampled randomly.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A weight of 1.0007-1.0015 g test item was weighed and directly added in 10 L test water to get the nominal concentration of 100 mg/L. The solution was stirred with magnetic stirrer using a stirring rate at about 200 rpm about 24 hours under the sealed condition and no headspace.12.5, 25.0, 50.0, 100 and 200 mL were diluted to 2L with test water to get the nominal concentration of 0.625, 1.25, 2.50, 5.00 and 10.0 mg/L, respectively. About 120 mL of each solution was dispensed into each test vessel to make the bottle have no headspace. The test bottle was sealed by stopper to keep a closed system.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: Daphnia magna Straus
- Age at study initiation (mean and range, SD): less than 24 hours old and not the first brood progeny
- Method of breeding:
- Source: Daphnia of stock culture were originally obtained from Applying Chemistry Department Pesticide Environmental Evaluation Laboratory of China Agricultural University. The test Daphnia were derived from a same healthy pre-cultured population
- Age of parental stock (mean and range, SD):
- Feeding during test
- Food type: Concentrated freshwater green algae(Chlorella sp and Desmodesmus subspicatus) suspension
- Amount: 50 μL, 0.1~0.2 mg C/Daphnia/day


ACCLIMATION
- Acclimation period: not specified
- Acclimation conditions: same as test
- Type and amount of food: Concentrated freshwater green algae(Chlorella sp and Desmodesmus subspicatus) suspension, 0.1~0.2 mg C/Daphnia/day
- Health during acclimation (any mortality observed): no signs of stress such as high mortality, presence of males and ephippia, delay in the production of the first brood, discoloured animals
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
170-190 mg/L (as CaCO3).
Test temperature:
19.5 - 21.0 °C
pH:
7.97-8.89
Dissolved oxygen:
8.28-9.37 mg/L
Nominal and measured concentrations:
Nominal: 0.625, 1.25, 2.5, 5.0 and 10 mg/L
Measured: 0.012, 0.023, 0.046, 0.093 and 0.204 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 120 mL glass bottle
- Type: closed
- Test solution volume: About 120 mL per vessel
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): three times a week, on every Monday, Wednesday and Friday.
- 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: Tap water treated with Aquapro Pure Water System
- Total organic carbon: < 2 mg/L
- Characteristics of Test Water: See "Characteristics of Test Water Quality" in attached backgroud material.
- Culture medium different from test medium: no
- Intervals of water quality measurement: every three months

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hr light : 8 hr dark
- Light intensity: 1000 ~ 1500 lux for cool white light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Offspring:
Offspring produced by each parent animal were removed and counted daily from the appearance of the first brood to prevent them consuming food intended for the parent.The number of living offspring and the presence of aborted eggs or dead offspring were recorded daily.
- Mortality: Mortality among the parent animals were recorded daily.
- Other parameters: Reproductive output per surviving parent animal, i.e. number of living offspring produced during the test per parent, were recorded. The length of the living parent animals (i.e. body length excluding the anal spine) at the end of the test were measured.
Other parameters measured or calculated include: time to production of first brood, total juveniles of each parent, total broods of each parent.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 3, 10, 30 and 100 mg/L (nominal concentration) and a control group
- Results used to determine the conditions for the definitive study: At 3 mg/L test group, the number of juveniles from the parent animals was significantly different in comparison with the control (p<0.05), and 10 mg/L test group, the number of juveniles from the parent animals was highly significantly different in comparison with the control (p<0.01), whereas there were several survival juveniles at 30 mg/L test group during 21 test days.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.023 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}
Details on results:
- Mortality:
During the 21 days exposure period, there was no death in the control group and in the 0.625, 1.25, 2.5 mg/L treatment group, 2 daphnids died in each treatment group of 5.00mg/L. All daphnids in the 10 mg/L treatment group were found dead after 21 days. The treatment group of 10 mg/L showed a significant difference compared to control group.

- Total living offspring per parent:
The total number of living offspring decreased with increasing test concentration. There was no significant difference for total living offspring per parent between the control and the test groups of 0.625 mg/L and 1.25 mg/L (Student´s T-test, P >0.05).There was a significant difference or highly significant difference for total living offspring between the control and the test groups of 2.5 mg/L, 5.0 mg/L and10 mg/L (Student´s T-test, P<0.05 or P<0.01).
For the control group, the coefficient of variation around the mean number of living offspring produced per parent animal at the end of the test was 12.2%, which was≤ 25%.

- First brood time:
In the control and all test groups, the first brood began on day 7 or 8 or 9. There was extremely significant difference for the first brood time between test groups of 10 mg/L and control (Student´s T-test, P<0.001).

- Length of the living parent animals:
There was significant difference for body length of parents between control and test group of 5.0 mg/L (Student T-test, P<0.05).

- Total number of broods:
No significant difference was found for the total number broods per parent between the control and the test groups of 0.625 mg/L, 1.25, 2.5 and 5.0 mg/L (Student´s T-test, P>0.05). There was an extremely significant difference for the total number of broods per parent between the control and the test group of 10 mg/L (Student´s T-test, P<0.001).

- Verification of Test Concentration:
Because this test item is easy to volatilize, the sample can’t be pretreated by concentration method. So far, there is no better method to analyze the test item but HPLC method. The test concentrations are too low that is under the LOQ. Therefore, the measured concentration of lower concentrations was calculated by the higher concentration.
The results showed that for the test solutions of 5.0 and 10 mg/L, the mean determined concentrations in fresh media were 0.097 and 0.219 mg/L. The mean determined concentrations in old media were 0.088 and 0.189 mg/L. The overall mean determined concentrations were 0.093 and 0.204 mg/L.
For the test solutions of 0.625, 1.25 and 2.50 mg/L, the measured concentrations were too low to be detected and the measured concentration was calculated by the measured concentration of 5.0 mg/L. The calculated concentrations were 0.012, 0.023 and 0.046 mg/L for the test solutions of 0.625, 1.25 and 2.50 mg/L.
Reported statistics and error estimates:
In summary, when NOEC and LOEC are being used to express the effects, it is recommended to calculate the effect on reproduction using both response variables:
• As the total number of living offspring produced per parent animal which does not die accidentally or inadvertently during the test and;
• As the number of living offspring produced per surviving parental animal;
Then to use as the final result, the NOEC, LOEC were determined by using the first of these two response variables.
Total number of living offspring per parent, body length of parent, maternal mortality, first brood time, total number of broods per parent were statistically analyzed. To obtain the no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC), a comparison of treatments to the control was calculated by using Student t-test with 5% significance level.

The 21-day NOEC and LOEC for all the parameters based on the measured concentration were as below:

Parameter

NOEC

(Measured concentration)

LOEC

(Measured concentration)

Total number of living offspring per parent

0.023 mg/L

0.046 mg/L

Body length of parent

0.046 mg/L

0.093 mg/L

Mortality parent

0.093 mg/L

0.204 mg/L

First brood time

0.093 mg/L

0.204 mg/L

Total number of broods per parent

0.093 mg/L

0.204 mg/L

Validity criteria fulfilled:
yes
Conclusions:
The 21-day NOEC and LOEC for Total number of living offspring per parent based on the measured concentration was 0.023 and 0.046 mg/L, respectively.
Executive summary:

A Daphnia magna Reproduction Test of test item was conducted over a 21 days exposure period according to OECD 211.

Based on the results of the preliminary test, the definitive test was carried out at concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L (nominal concentration) with a semi-static system with solution renewed at three times a week, on every Monday, Wednesday and Friday. A concurrent blank control was performed in parallel. 10 daphnids individually held were used for each test group and the control group.

The 21-day NOEC and LOEC for Total number of living offspring per parent based on the measured concentration was 0.023 and 0.046 mg/L, respectively.

Description of key information

A Daphnia magna Reproduction Test was conducted over a 21 days exposure period according to OECD 211.

The 21-day NOEC and LOEC for Total number of living offspring per parent based on the measured concentration was 0.023 and 0.046 mg/L, respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.023 mg/L

Additional information

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