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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:
1999-12-07 to 21-04-2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.400, 0.200, 0.100 0.0500, 0.0250 mg/L (nominal)
- Sampling method: Samples were taken 6 times (3 times repeated before and after changing the water) during the exposure period for all test plots (one per test vessel), and after removing food from the samples before changing the water by centrifugation.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the necessary amount of test substance to achieve a concentration of 10,000 mg/L was dissolved in DMSO. The test solution was
prepared by adding a necessary amount of the undiluted test solution to the dilution water put in a preparation vessel while stirring .
- Controls: 10 replicates each of dilution water and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethyl sulfoxide (DMSO)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 100 µl/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea ,Ohmijinko (japanese), (Daphnia magna)
- Strain/clone: Clone A
- Source: Sheffield University. Sheffield S10 2Uq, United Kingdom
- Feeding during test
- Food type: Chlorella vulgaris
- Amount: 0.1-0.2 mg C (organic carbon) per day per Daphnia

ACCLIMATION
- Acclimation period: 2000.01.03-2000.31.03
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: Chlorella vulgaris. 0.1-0.2 mg C per day per Daphnia
- Health during acclimation (any mortality observed): Death rate was 5 % or less

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
37.0-48.4 mg/L as CaCO3
Test temperature:
20.0-20.5 °C
pH:
7.4-7.9
Dissolved oxygen:
8.3-8.8 mg/L
Nominal and measured concentrations:
0.0250, 0.0500, 0.100, 0.200, 0.400 mg/L (nominal)
0.187, 0.0331, 0.06, 0.117, 0.247 mg/L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 100 mL beaker:
- Renewal rate of test solution (frequency/flow rate): once per day
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Kurume City tap water
- Metals: Tin, 0.02 mg/L
- Chlorine: 0.02 mg/L or lower
- Alkalinity: 27.5 mg/L
- Ca/Mg ratio: 2.62
- Conductivity: 146 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Water quality test performed on Februrary 8, 2000

OTHER TEST CONDITIONS
- Photoperiod: 16 hours
- Light intensity: 1200 lux or lower

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Reproduction and mortality. Every day, the number of surviving and immobilized Daphnia were recorded. Every day, the number of live and dead juveniles, occurences of aborted eggs and diapause eggs were recorded.

VEHICLE CONTROL PERFORMED: Yes

RANGE-FINDING STUDY: Yes, but results were not reported.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.231 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: 95% CL: 0.209 - 0.265 mg/L
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
0.195 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: 95% CL: 0.177 - 0.221 mg/L
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
0.195 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: mortality of parental Daphnia
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
0.208 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: mortality oft parental Daphnia
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.177 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: average cumulative number of brood
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.247 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: average cumulative number of brood
Details on results:
- Mortality of parent animals: No mortalities were observed in any of the control plots. Among all test plots, only the concentration at 0.247 mg/L showed any mortalities. On day 4, seven mortalities were observed and then one further mortality occured on day 17, bringing the total number of mortalities to 8 after 21 days. The other concentration plots did not show any deaths.
- No. of offspring produced per day per female: See table 1
- Time to first brood release or time to hatch: The mean brood release time for all concentrations and controls was 8 days, except the test concentration at 0.267 mg/L, which took on average 8.7 days for brood production.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
48-hour median immobilization concentration (EiC50) by the reference material (potassium dichromate, reagent chemical of highest quality, Wako Pure Chemical Industries, Ltd.) = 0.141 mg/L [EiC50 after January 1998 in Kurume Business Office: Within the range of 0.128~0.234 mg/L (n = 5).]
Reported statistics and error estimates:
Calculation of EC50:
EC50 was calculated by the moving average method. The calculation was made by applying the maximum average cumulative number of brood (rounding up digits to the right of the decimal point) over all the test plots to the number of specimens under testing, and the average cumulative number of brood in each concentration plot to the number of survivers. EC50 of the 14th and 21st days were calculated, and their 95 % confidence limits are also shown.
Calculation of maximum no observed effect concentration (NOEC) and lowest observed effective concentration (LOEC):
The cumulative number of brood per one parent for each test vessel was calculated, after performing homoscedastic testing by the Bartlett method.
Significant difference between each concentration plot and the solvent control plot was determined by one-way analysis of variance and Dunnett's multiple comparison method.
Also, the significant difference between each concentration plot and the control plot for the number of dead parental Daphnia was determined by the Kruskal-Wallis rank sum test and the nonparametric multiple comparison method (Dunnett). The nonparametric multiple comparison method (Scheffé) was used for the primiparous day. NOEC and LOEC were determined including the states of observed parents and produced juveniles in this result.

Table 1: Mean cumulative number of juveniles produced per adult (Σ F1/P) during exposure to di-α-cumyl peroxide and average juvenile produced per day over 21 days.

Measured concentration

(mg/L)

Exposure time (days)

8

9

10

11

12

13

14

15

Control

13.9

NC

NC

40.3

NC

NC

71.2

74.1

Solvent control

13.5

NC

NC

35.9

37.1

NC

69.4

72.6

0.0187

14.5

NC

NC

42.2

NC

NC

75.0

75.6

0.0331

14.4

NC

NC

36.9

37.6

NC

69.8

NC

0.600

13.3

NC

NC

38.3

NC

NC

72.3

72.6

0.117

11.2

NC

NC

35.2

NC

NC

65.9

66.1

0.247

2.5

4.5

NC

14.0

23.5

NC

NC

39.0

* table begins on day 8 when the first juveniles appeared

*NC = no change from the previous day´s value.

Table 1 continued:

Measured concentration (mg/L)

Exposure time (days)

Average number produced per day

16

17

18

19

20

21

Control

74.1

NC

103

NC

NC

128

6.10

Solvent control

72.6

NC

104

NC

NC

129

6.14

0.0187

75.6

NC

108

NC

NC

134

6.38

0.0331

72.0

NC

107

NC

NC

131

6.24

0.0600

75.8

NC

106

NC

NC

133

6.33

0.117

68.3

NC

96.4

NC

NC

120

5.71

0.247

39.0

NC

52.0

53.0

NC

60.0

2.86

The average cumulative number of brood per parental Daphnia in the 0.247 mg/L plot was 60, showing a significant difference compared with that in the solvent control plot. The average cumulative numbers of brood per parental Daphnia in the 0.117~0.0187 mg/L plots were 120, 133, 131, and 134, respectively, almost the same as those in the solvent control plot and the control.

Size and state of parental Daphnia

During the exposure period, body color brightening and growth inhibition were observed in the 0.247 mg/L plot compared with the control plot, besides lethargy, immobility, and activity drop were observed. No symptoms were observed in the other concentration plots.

Occurrence of diapause eggs, etc.

Throughout the entire exposure period, no occurrence of diapause eggs was observed in any test plot.

Dead larvae were observed in all the test plots, and the 0.247 mg/L plot had somewhat more than the control plot. Fallen eggs were also observed in all of the test plots, the 0.117 mg/L showed a statistically significant difference.

Validity criteria fulfilled:
yes
Conclusions:
The 21-day-chronic toxicity of dicumylperoxide to Daphnia magna was studied under static renewal conditions. The parental 21-day LC50 based on mortality was 0.195 mg/L, the 21-day EC50 for reproduction was 0.231 mg/L. The 21-day NOEC based on average cumulative number of brood was 0.117 mg/L, the 21-day LOEC was 0.247 mg/L. Sublethal effects were observed in the 0.247 mg/L plot.
Production of offspring in the treated groups indicated that dicumylperoxide had an effect on reproduction at concentrations greater than 0.117 mg/L. The most sensitive end point was reproduction.
Executive summary:

The 21-day-chronic toxicity of dicumylperoxide to Daphnia magna was studied under static renewal conditions. Daphnids were exposed to control, solvent control, and test chemical at measured concentrations of 0.187, 0.0331, 0.0600, 0.117, 0.247 mg/L.  The parental 21-day LC50 based on mortality was 0.195 mg/L, the 21-day EC50 for reproduction was 0.231 mg/L. The 21-day NOEC based on average cumulative number of brood was 0.117 mg/L, the 21-day LOEC was 0.247 mg/L.  The sublethal effects included were body colour brightening and growth inhibition in the 0.247 mg/L plot compared with the control plot, besides lethargy, immobility, and activity drop were observed. No symptoms were observed in the other concentration plots. Dead larvae were observed in all the test plots, and the 0.247 mg/L plot had somewhat more than the control plot. Fallen eggs were also observed in all of the test plots, the 0.117 mg/L showed a statistically significant difference.

Production of offspring in the treated groups indicates that dicumylperoxide had an effect on the reproduction at concentrations greater than 0.117 mg/L.  The most sensitive endpoint was reproduction.

This study is classified as reliable without restriction and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates.

Results synopsis

Test organism age: First instar

Test type (static renewal): OECD guideline 211 (Daphnia magna reproduction test)

21-day EC50 (reproduction): 0.231 mg/L (95% CL: 0.209–0.265 mg/L)

21-day LC50 (parental mortality): 0.195 mg/L

21-day NOEC (average cumulative number of brood): 0.117 mg/L

21-day LOEC (average cumulative number of brood): 0.247 mg/L

Endpoint(s) effected: Reproduction, parental mortality

Description of key information

21-day NOEC (average cumulative number of brood): 0.117 mg/L

Endpoint(s) effected: Reproduction

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.117 mg/L

Additional information

The 21-day chronic toxicity of dicumyl peroxide to Daphnia magna was studied under static renewal conditions. The parental 21-day LC50 based on mortality was 0.195 mg/L, the 21-day EC50 for reproduction was 0.231 mg/L. The 21-day NOEC based on average cumulative number of brood was 0.117 mg/L, the 21-day LOEC was 0.247 mg/L. Sublethal effects were observed in the 0.247 mg/L plot (body colour brightening and growth inhibition, besides lethargy, immobility, and activity drop). No symptoms were observed in the other concentration plots. Dead larvae were observed in all the test plots, and the 0.247 mg/L plot had somewhat more than the control plot. Fallen eggs were also observed in all of the test plots, the 0.117 mg/L showed a statistically significant difference. Production of offspring in the treated groups indicated that dicumyl peroxide had an effect on the reproduction at concentrations greater than 0.117 mg/L. The most sensitive end point was reproduction.