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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:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Testing was based upon the OECD chemical testing guidelines No. 202 “Order Cladocera, Acute Immobilization Testing and Reproduction Testing” (1984). This is a former version of the OECD 211 guidelines (Daphnia magna reproduction test). Since n-nitrosodiphenylamine is not soluble, a vehicle was used. Although not recommended, this is not prohibited by the OECD 211 guidelines. This study is GLP compliant.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
The test is almost identical to OECD 211. The main difference is that daphnia parents are grouped instead of being individualised. Here, there are 40 daphnia per concentration while there are only 10 per concentration in the 211 OECD guidelines.
Principles of method if other than guideline:
Exposure Type: Semi-static method (Total volume of test fluid replaced every 2 days after start of exposure)
Exposure Period: 21 days
Test fluid volume: 800 mL (The test fluid volume was increased in order to maintain the dissolved oxygen concentration)
Number of replicates: 4 per concentration
Number of organisms: 40 per concentration (10 per replicate)
Test water temperature: 20±1°C
Illumination: Indoor light, 16 hours lit/8 hours dark
Feed: Chlorella vulgaris
Feeding Volume: 0.1~0.2 mgC (organic carbon content)/day per Daphnia Magna

The test is almost identical to OECD 211. The main difference is that daphnia parents are grouped instead of being individualised. Here, there are 40 daphnia per concentration while there are only 10 per concentration in the 211 OECD guidelines.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations (nominal): 0.1 ; 0.32 ; 1 ; 3.2 ; 10 mg/L (+control + solvent control)
- Sampling method:
- Sample storage conditions before analysis: not detailed
Vehicle:
yes
Details on test solutions:
400 mg of the tested substance was weighed out and dissolved into 2000 mg dimethylformamide (DMF) and 1200 mg HCO-30 by stirring. This was then diluted with purified water to a set volume of 500 mL, making a stock solution with a test substance concentration of 800 mg/L and 4000 mg/L DMF and 2400 mg/L HCO-30. At the same time, a 6400 mg/L adjuvant stock solution not containing the test substance (DMF 4000 mg/L, HCO-30 2400 mg/L) was prepared.
For each concentration to be tested, 800 mL of purified water were placed into a 1-L glass beaker, and after adding the above test substance stock solution according to the required final concentration, the adjuvant stock solution was added so that the adjuvant concentration was constant among each beaker (80 mg/L). The control groups used the dilution water or the solvent containing dilution water only.
Test organisms (species):
Daphnia magna
Details on test organisms:
This variety was obtained from the National Institute of Environmental Studies (Japan) on July 18, 1995, and has been successively cultured at this laboratory.

Daphnia Magna Culturing Method to Obtain Tested Larva
Female adults holding larva that appeared healthy and sufficiently large on gross examination were selected in the succession, transferred to a separately prepared beaker, and the next day the larvae produced were separated to a different beaker. These larvae (born January 29, 1997) were treated as the parents of the tested Daphnia Magna, and where raised under the following conditions. Once reaching maturity and producing larvae, these larvae were removed 2 times per week. At the 2nd week, female adults with larvae in their brood chambers were selected from a batch that had a mortality rate of 0% over the previous 2 weeks and that had not produced dormant eggs or males, and the larvae produced the next day (within 24 hours) were used in the test (Exposure started on February 12, 1997).

Domestication Conditions
1) Culturing water: Dilution water
2) Culture Concentration: When Immature: 35 or 70/3L culture water
When adult: 35/3L culture water
3) Water Temperature: 20±1°C
4) Illumination: Indoor light, 16L:8D
5) Feed: Chlorella vulgaris
6) Feed volume: 0.1-0.2 mg C (organic carbon content) per day per Daphnia magna
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None.
Hardness:
62 mg/L CaCO3
Test temperature:
19.4-20.3 (min-max)
pH:
7.5-8.2 (min-max)
Dissolved oxygen:
6.7-8.6 (min-max)
Salinity:
10 mg/L Sodium
Nominal and measured concentrations:
- Nominal: 0 ; 0.1 ; 0.32 ; 1 ; 3.2 ; 10 mg/L
- Measured (time weighted): 0.075 ; 0.26 ; 0.856 ; 2.92 ; 10.1* mg/L

*Time weighted mean for 3 days, since all parents were dead after 3 days exposure
Details on test conditions:
TEST SYSTEM
- Test vessel: 1-L glass beaker
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 1L
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel:
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: De-chlorinated water (Yokohama municipal tap water processed with activated charcoal, removing residual chlorine, etc., and thereafter fully aerated)
- Mercury: <0.0001 mg/L
- Copper: 0.0007 mg/L
- Cadmium: <0.001 mg/L
- Zinc: 0.01 mg/L
- Lead: <0.01 mg/L
- Aluminium: 0.02 mg/L
- Nickel: 0.01 mg/L
- Chromium: <0.005 mg/L
- Manganese: <0.1 mg/L
- Tin: <0.01 mg/L
- Iron: <0.12 mg/L
- Cyanide: <0.001 mg/L
- Bromide ion: <0.1 mg/L
- Fluoride: 0.12 mg/L
- Sulfide ion: <0.03 mg/L
- Ammonium: <0.1 mg/L
- Arsenic: <0.005 mg/L
- Selenium: <0.005 mg/L
- Pesticides: not assessed
- Chlorine: <0.01 mg/L
- Alkalinity: 52 mg/L
- Ca/mg ratio: 3.3
- Conductivity: 180 µS/cm
- Salinity: 10 mg/L sodium
- Culture medium different from test medium: no
- Intervals of water quality measurement: not mentioned

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16L:8D
- Light intensity: not mentioned

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Parental survival (daily checked) LC50, NOEC, LOEC
- Number of neonates (daily checked) NOEC, LOEC

VEHICLE CONTROL PERFORMED: yes (DMF + HCO-30)

RANGE-FINDING STUDY: none
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
4.62 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: CI95 3.84-5.71
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
4.42 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: CI95 = 3.68-5.43
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.075 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.26 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Number of Parent Daphnia Magna Deaths and Mortality Rate
The mortality rate for parent Daphnia Magna in the control plot and the adjuvant control group was 3% in both upon conclusion of exposure, satisfying the standard of 20% or less, which is a test establishment condition. The mortality rate for parent Daphnia Magna at the conclusion of exposure was 8% in the 0.100 mg/L plot, 0% in the 0.320 mg/L plot, 3% in the 1.00 mg/L, 8% in the 3.20 mg/L plot, and 100% in the 10.0 mg/L plot.

First Hatching Date
The first hatching dates of the parent Daphnia magna in the control group were 7 and 8 days according to the replicate after start of exposure, while in the adjuvant control group it was after 7 days in all replicates, and satisfied the standards of within 9 days, which is a test establishment conditions. The first hatching date of the parent Daphnia Magna in each concentration replicate was 7 days after start of exposure in the 0.100 and 0.320 mg/L groups, and after 7 to 8 days in the 1.00 mg/L group. It was 10 and 14 days after the start of exposure in the 3.20 mg/L group. In addition, no juveniles were hatched in the 10.0 mg/L plot.

Average Number of Cumulative Juveniles
The average number of cumulative juveniles per parent Daphnia magna at 21 days in the control group and the adjuvant control group was 71 and 76, respectively, satisfying the standard of 40 for the average number of cumulative juveniles, which was a test establishment condition.
The average number of cumulative juveniles per parent Daphnia magna in each of the concentration groups was 80 in the 0.100 mg/L group, 62 in the 0.320 mg/L group, 57 in the 1.00 mg/L group, and 5 in the 3.20 mg/L group.

Size and Condition of the Parent Daphnia Magna
In the 1.00 mg/L group, the parent Daphnia Magna appeared to be small compared to the control group at 3 and 4 days after start of exposure. In the 3.20 mg/L group the parent Daphnia Magna were small compared to the control group from the 2nd to the 13th day after the start of exposure, and furthermore they appeared quite small from the 14th day until the end of exposure. In the 10.0 mg/L group, which was the maximum concentration group, the parent Daphnia Magna appeared small compared to the control group from 2 days after the start of exposure, and all parent Daphnia Magna were dead 3 days after starting.
In the other concentration groups, no difference in size of the parent Daphnia Magna was seen compared to the control plot throughout the entire exposure period.

Generation of Dormant Eggs, etc.
The occurrence of dormant eggs was not seen in the control plot as well as all concentration plots throughout the entire exposure period.
Results with reference substance (positive control):
The average number of cumulative juveniles per parent Daphnia magna at 21 days in the control group and the adjuvant control group was 71 and 76, respectively, satisfying the standard of 40 for the average number of cumulative juveniles, which was a test establishment condition.
Reported statistics and error estimates:
The Median Lethal Concentrations (LC50) and the 95% reliability limits for the 14th and 21st day of exposure were calculated by the moving average method from the number of dead parent Daphnia magna and the number of test specimens (40) in each concentration group.
The cumulative juveniles per parent for each test container was calculated, and the presence of a significant difference between each concentration group and the adjuvant control group was derived by the Dunnett multiple comparison method (a=0.05, bilateral), and the maximum concentration where no significant difference was seen (minimum effect concentration: NOECr) as well as the minimum concentration where a significant difference can be seen (LOECr) were determined. In the statistical analysis, Yukms Software Statlight “#4 multi-group comparison} (Yukms Corp., Tokyo) was used.

Results of the experiment after 21 days exposure. Daphnia from the 10 mg/L group were all dead after 3 days exposure.

 

Control

Solvent control

0.1

0.32

1

3.2

10

Parent mortality (%)

3

3

8

0

3

8

100

Time to 1st brood (d)

7.25

7

7

7

7.75

12

-

Neonates per parent

71

76.1

80.3

61.7

56.6

5.4

0

Validity criteria fulfilled:
yes
Conclusions:
Daphnia parents were exposed to a graded series of nitrosodiphenylamine concentrations for 21 days. The NOEC and LOEC on juveniles production were 0.075 and 0.23 mg/L respectively. Estimated parental LC50 after 14 and 21 days exposure were 4.42 and 4.62 mg/L respectively.
Executive summary:

Daphnia parents were exposed in quadruplicate to a graded series of n-nitrosodiphenylamine concentrations for 21 days, according to OECD 202 guidelines (1984), equivalent to actual OECD 211 guidelines (2012), with minor differences. Nominal concentrations were 0.1; 0.32 ; 1 ; 3.2 and 10 mg/L. Measured concentrations (time weighted) were 0.075 ; 0.26 ; 0.856 ; 2.92 and 10.1 mg/L. Replicates were daily checked for parental immobilization and neonates production. After 21 days, the NOEC and LOEC on juveniles production were 0.075 and 0.23 mg/L respectively. Estimated parental LC50 after 14 and 21 days exposure were 4.42 and 4.62 mg/L respectively.

Description of key information

EC10 of inhibition of neonates production by female Daphnia magna for 21 days was 0.075 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.075 mg/L

Additional information