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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 April 04, 2003 to July 31, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been conducted according to the appropriate OECD guideline, under GLP and analytical monitoring was carried out.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes
Remarks:
(Certificate is not included in the study report.)
Analytical monitoring:
yes
Details on sampling:
A 0.75 mL of the test solution before and after water renewala was collected from a test vessel each at all test areas for three times during the exposure period. The equivalent amount of acetone was added, mixed and the solutions were analyzed by GC.

Vehicle:
no
Details on test solutions:
Test substance solution;
For the dilution water at preparation of the test solution, aeration was performed before preparation and temperature was set to be 20 ± 1°C in the thermostat bath.
Preparation of the stock solution of the test substance;
The test substance of 2000 mg was sampled and added in the purified water and made to constant volume of 20 mL with purified water to give 10000 mg/L test substance concentration. This stock solution was prepared at the start of exposure and every 6 or 7 days and stored in refrigerated and dark place. This solution was stable for 13 days.
The stock solution containing the test substance was collected in the appropriate amount to the test concentrations, quantified to 1.0 L with the dilution water. 80 mL of this solution was put into 10 test vessels each per test concentration.

Control area;
Only dilution water was used for the control area.
Dilution water;
Elendt M4, the prepared water described in OECD Guideline for testing of Chemicals No.211, "Daphnia sp., Reproduction Test", was used.
Test organisms (species):
Daphnia magna
Details on test organisms:
1) Japanese name: Oomijinko
2) Scientific name: Daphnia magna
3) Obtained from: National Institute for Environmental Studies, Environmental Agency (current, National Institute for Environmental Studies, independent administrative agency)
4) Date of obtainment: July 18, 1995
5) Sensitivity: An acute immobilization test is periodically (about every six months) performed with the standard material (potassium bichromate, special grade chemicals) to examine the sensitivity of the test living organism. 48-hour 50% immobilization concentration (EC50) since June in 1998 is as follows; Mean ± Standard deviation = 0.75 ± 0.17 mg/L, n = 10 (Minimum value to maximum value = 0.57 to 1.02 mg/L)
6) Developing stage: female juveniles (in 24 hours after being produced)
7) Breeding conditions for parental Daphnia to obtain juveniles for testing:
Breeding water: dilution water
Breeding density: one organism/80 mL (25 organisms/2L) or less
Breeding vessel: 2 L glass vessel
Water temperature: 20 ± 1°C
Dissolved oxygen concentration: 60% or more of the saturation concentration
pH: 6.7 to 8.5
Lighting: room light, 16/8 hours light (800 lux or less)/dark cycle
Rearing period: from March 13, 2003 to April 10, 2003
The mortality of the parent Daphnia during 2 weeks before the start of exposure: 0%
Occurrence of ephippia and male: absent
Type of feed: Chlorella vulgaris (monocellular green alga) (alga culture solution was centrifuged, replaced with dilution water and used.)
Amount of food: 0.2 mg C (content of organic carbon)/animal/day
Renewal of the rearing water: renewed periodically (3 times/week). The juveniles are removed as much as possible every day.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
(as CaCO3, unit: mg/L)
Control; 270 (0 d), 270 (1 d), 270 (7 d), 255 (8 d), 250 (14 d), 250 (15 d), 250 (20 d) and 250 (21d), (Min.: 250, Max. 270)
Test solution (95.1 mg/L*); 270 (0 d), 270 (1 d), 260 (7 d), 255 (8 d), 255 (14 d), 250 (15 d), 250 (20 d) and 245 (21d), (Min.: 245, Max. 270)
*: Time-weighted mean measured concentration
Test temperature:
(unit: °C)
Control; 20.0 (0 d), 19.7 (1 d), 19.8 (7 d), 20.4 (8 d), 20.3 (14 d), 20.2 (15 d), 20.1 (20 d) and 19.7 (21d), (Min.: 19.7, Max. 20.4)
Test solution (95.1 mg/L*); 20.2 (0 d), 19.7 (1 d), 19.8 (7 d), 20.2 (8 d), 20.2 (14 d), 20.2 (15 d), 20.1 (20 d) and 19.6 (21d), (Min.: 19.6, Max. 20.2)
*: Time-weighted mean measured concentration
pH:
Control; 8.2 (0 d), 8.2 (1 d), 8.2 (7 d), 7.7 (8 d), 8.2 (14 d), 7.7 (15 d), 8.3 (20 d) and 7.6 (21d), (Min.: 7.6, Max. 8.3)
Test solution (95.1 mg/L*); 8.2 (0 d), 8.1 (1 d), 8.2 (7 d), 7.6 (8 d), 8.2 (14 d), 7.7 (15 d), 8.3 (20 d) and 7.6 (21d), (Min.: 7.6, Max. 8.3)
*: Time-weighted mean measured concentration
Dissolved oxygen:
(unit: mg/L)
Control; 8.8 (0 d), 8.6 (1 d), 8.7 (7 d), 8.2 (8 d), 8.8 (14 d), 7.9 (15 d), 8.8 (20 d) and 7.3 (21d), (Min.: 7.3, Max. 8.8)
Test solution (95.1 mg/L*); 8.8 (0 d), 8.5 (1 d), 8.8 (7 d), 8.1 (8 d), 8.8 (14 d), 7.8 (15 d), 8.8 (20 d) and 7.3 (21d), (Min.: 7.3, Max. 8.8)
*: Time-weighted mean measured concentration
Salinity:
No description
Nominal and measured concentrations:
Test concentration;
The test concentration in this study was determined as 100 mg/L (nominal value, the limiting test).
Measured test substance concentration; see Table 1
Details on test conditions:
1) Exposure method: Semi-static (the total amount of the test solution was renewed every day). (The water surface was covered with Teflon sheet.)
2) Exposure duration: 21 days
3) Amount of the test solution: 80 mL/vessel
4) Replicate number: 10 vessels/test area
5) The number of the test living organisms: 10 organisms/test area (one organism/vessel)
6) Test temperature: 20 ± 1°C
7) Dissolved oxygen concentration: 60% or more of the saturation concentration. Aeration was not performed during the exposure period.
8) pH: pH of the test solution was not adjusted.
9) Hardness: about 250 mg/L (converted to CaCO3)
10) Lighting: room light, 16/8 hours light (800 lux or less)/dark cycle
11) Feeding: type of feed: Chlorella vulgaris (monocellular green alga), (alga culture solution was centrifuged, replaced with dilution water and used.)
12) Feeding dosage: 0.15 mg C (content of organic carbon) / organism / day
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 95.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: mortality and reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 95.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: mortality and reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 95.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: mortality and reproduction
Remarks on result:
other: 95% CL cannot be calculated
Details on results:
(1) Concentration of the test substance in the test solution;
The concentration of the test substance in the test solution was measured before and after water renewal for three times during the exposure period.
As the results of the analysis of the test solution, the mean percentage of the measured value to the nominal value was 95%. (see. Table 1)
(2) Number of dead parental Daphnia magna and its mortality;
The mortality of parental Daphnia magna in the control area was 0% at completion of exposure, the criteria of 20% or less for establishment of the study was fulfilled. The mortality in the 100 mg/L concentration area was 0% at completion of exposure.
(3) Date of the first brood production;
The date of the first brood production of parental Daphnia magna in the all test vessels of control area was 8-days after the start of exposure which considered to be within a normal range.
The date of the first brood production was 8-days after starting exposure in the all test vessels of 100 mg/L concentration area.
(4) Mean cumulative number of juveniles produced;
The mean cumulative number of juveniles produced per adult alive in the control area for 21 days was 115 juveniles, the criteria of 60 or more juveniles for establishment of the study was fulfilled.
The mean cumulative number of juveniles produced per adult alive in the 100 mg/L concentration area for 21 days was 114 juveniles.
(5) Occurrence of ephippia etc.;
No occurrence of ephippia was recognized in all test areas during the exposure period.
(6) 50% Lethal Concentration of parental Daphnia (EC50);
21-day LC50: >95.1 mg/L (95%CI, cannot be calculated)
(7) 50% Reproduction inhibition concentration (EC50);
21-day LC50: >95.1 mg/L (95%CI, cannot be calculated)

Table 2 : Mean cumulative number of juveniles produced per adult alive for 21 days

Nominal

Con. (mg/L)

Measured

Con. (mg/L)*

Day

6

7

8

9

10

11

12

13

Control

---

0

0

9.9

9.9

9.9

33.7

33.7

33.7

100

95.1

0

0

7.7

7.7

7.7

27.5

29.1

29.2

Nominal

Con. (mg/L)

Measured

Con. (mg/L)*

Day

14

15

16

17

18

19

20

21

Control

---

41.0

58.6

58.6

58.6

88.6

88.6

88.6

115.3

100

95.1

43.7

55.7

55.7

58.5

85.7

85.7

85.7

113.9

*: Time-weighted mean measure concentration
Validity criteria fulfilled:
yes
Conclusions:
A 21 d NOEC value of ≥95 mg/l has been determined for the effects of the test substance on mortality of the freshwater invertebrate Daphnia magna. In the same study a 21 d EC50 value of >95 mg/l was determined. The results are based on geometric mean measured concentration.

Description of key information

21 d, NOEC ≥95.1 mg/L, Daphnia magna, measured.

21 d, EC50 >95.1 mg/L, Daphnia magna, measured.

Key value for chemical safety assessment

Additional information

A 21 d NOEC value of ≥95 mg/l has been determined for the effects of the test substance on mortality of the freshwater invertebrate Daphnia magna. In the same study a 21 d EC50 value of >95 mg/l was determined. The result are based on geometric mean measured concentration.

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