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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:
21 August 2002 to 11 September 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese MAFF Daphnid reproduction test (2-7-2-2)(12 Nousan No 8147)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Test solutions were measured 6 times during the exposure period (3 times iteration of every week measurement of fresh and spent solution). The fresh test solutions for measurement were sampled from preparation vessel in each exposure level. The spent test solutions were taken from the middle layer in one of 4 vessels in each exposure level, and then pretreated by centrifugation for 10 minutes at 3,000 rpm to remove food.
Vehicle:
no
Details on test solutions:
Desired amount of the test material which was calculated by its density (2.26 g/cm^3 (25 "C)] was measured and dissolved in dilution water to prepare the stock
solutions of 100 mg/L. The necessary amount of the stock solution was mixed with dilution water in a container for preparation and then the mixed solution was divided
into each test vessel. The preparation was separately performed for each exposure level.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Clone: Clone A
- Source: Daphnid stock originally from Univerity of Sheffield (UK). Parents cultured in Kurume Laboratories
- Age: Daphnids less than 24 hours old produced by 29 day old parents were used in the test.
- Food type: Unicellular green algae (Chlorella vulgaris)
- Amount: 0.1-0.2 mg C/daphnid/day
- Frequency: once a day
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
10-250 mg CaCO3/L
Test temperature:
21 ± 1°C
pH:
6-9 with no variation of more than 1.5 units during the exposure
Dissolved oxygen:
8.4-8.9 mg/L
Nominal and measured concentrations:
- Nominal concentrations: 0.020, 0.040, 0.08, 0.16 and 0.32 mg/L
- Mean measured concentrations: 0.0197, 0.0393, 0.0777, 0.156 and 0.279 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass vessel closed with cover (watch glass)
- Type: closed
- Renewal rate of test solution: Test solutions were renewed on a daily basis and the daphnids were transferred from the “old” to the “new” solutions.
- No. of organisms per vessel: 5 daphnids per vessel
- No. of vessels per concentration : 4 vessels per concentration
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
- Dilution water: Dechlorinated tap water

OTHER TEST CONDITIONS
- Photoperiod: 8 hours light and 16 hours dark
- Light intensity: Less than 1200 lux

EFFECT PARAMETERS MEASURED:
- Mortality of parent daphnids was observed on a daily basis. Size and appearance, swimming behaviour, determination of sex, presence of eggs in the brood pouch, ephippia and aborted broods were also recorded daily.
- From the onset of offspring production, the number of juveniles was counted on a daily basis and their condition was noted. The offspring were discarded after counting.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 1.0, 0.316, 0.100 and 0.0316 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.23 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
0.205 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
OBSERVATIONS ON DAPHNIDS
- Mortality of parent daphnid: The cumulative mortality of parent daphnid in the control at the end of exposure was 20.0 % and it met the validity of the test [that of the parent daphnid (female Daphnia) does not exceed 20 % at the end of the test in the control]. The cumulative mortality of parent daphnid in the 0.320, 0.160, 0.0800, 0.0400 and 0.0200 mg/L levels at the end of exposure were 20, 5, 10, 15 and 30 %, respectively. Since the overall mortalities of parent daphnid in the present test tend to be a little higher than the usual cases and no relationship between concentration and mortality was observed, it is thought that the mortality of 30 % in 0.0200 mg/L level was caused by biologically problematic factor of the test organisms. The concentration of 0.0200 mg/L is the lowest exposure level and it is not the level related to the adverse effect on reproduction. Therefore, it is thought that the mortality in 0.0200 mg/L level doesn't work against the final estimation. For the mortalities of parent daphnid in all exposure levels, no statistically significant difference was observed compared with the control.
- Time to first production of offspring: The time to first production of offspring in the control was 8 days and it was considered to be normal. The time to first production of offspring in the exposure levels ranging from 0.160 to 0.0200 mg/L level were also 8 days and no significant difference was found between the treatments and the control. Since the highest concentration of 0.320 mg/L was greater than EC50, the treatment level was not considered in the statistical analysis. However, the values in the highest exposure level of 0.320 mg/L were 9 to 15 days in each vessel, which mean value was 11.3 days, and thus the delay of the time was obviously recognised compared with that in the control.
- Mean cumulative number of live offspring: The mean cumulative number of live offspring per parent daphnid alive in the control during 21 days was 104 and it met the validity of the test [that is more than 60 at the end of the test]. The mean cumulative numbers of live offspring per parent daphnid alive in 0.160 to 0.0200 mg/L level were 100 to 136. The tendency of increasing the cumulative numbers of offspring was observed in 0.160 and 0.0800 mg/L level and these values were significantly different from those in the control. The mean cumulative numbers of live offspring per parent daphnid in 0.320 mg/L level was 0.1 and an adverse effect at the concentration on reproduction was obviously recognised. The reason that caused the reproductive increase observed in 0.160and 0.0800 mg/L level were unclear, however, it is considered that one of the reason might be based on hormesis.
- Size and condition of parent daphnid: Some appearances of light body colour since 14 days, growth inhibition since 16 days, attachment of chlorella to body since 18 days and immobilisation on 21 days were observed for a few parent daphnids in the control. In 0.0200 and 0.0400 mg/L level, a few parent daphnids showed the appearances of attachment of chlorella to body since 10 days, light body colour since 12 or 13 days, growth inhibition since 16 days and immobilisation since 19 days. Likewise, light body colour since 13 days and attachment of chlorella to body since 19 days were observed in 0.0800 mg/L level. No abnormal appearances were observed in 0.160 mg/L level. In 0.320 mg/L level, more numbers of parent daphnids than other levels showed the appearances of light body colour and change to white colour of eggs in brood pouch since 14 days, lethargic condition since 20days, and attachment of chlorella to body and immobilisation on 21 days. These conditions in 0.320 mg/L level were apparently considered as adverse effect.
- Occurrence of ephippium and so on: One ephippium was observed in 0.0200 mg/L on 15 days after the exposure. Dead offspring and aborted egg were observed in 0.320 and 0.160 mg/L levels.

LC50 FOR PARENT DAPHNID
- Both 14-day and 21-day LC50s for the parent daphnids were >0.320 mg/L.

EC50
- The 14-d EC50 was 0.205 mg/L and the 21-day EC50 was 0.230 mg/L. The reason that caused the reverse of 14-day and 21-day ECSO is unclear, but it is thought that the increase of offspring produced by hormesis after 14 days may get involved with it, and/or that the test material does not have a characteristic of chronic effect and by then it may be only within the range based on the numerical variation arising from the difference of the number of offspring until 14 days or 21 days.

LOEC AND NOEC FOR REPRODUCTION
- Regarding the mean cumulative number of live offspring per parent daphnid alive, a significant difference was recognized between 0.0800 and 0.160 mg/L levels and the control. However, the difference shown in these levels was based on an increase of offspring compared with the control. Since the effect was not reproductive impairment, it was estimated that LOEC was 0.320 mg/L where apparent inhibition of reproduction was observed, and then NOEC was 0.160 mg/L.

OBSERVATION AND MEASUREMENT OF TEST SOLUTION
- Condition of test solution: The test solutions in the exposure levels were clear and colourless at the preparation, and they turned to a pale green before the renewal due to unicellular green alga added as food.
- Water temperature, dissolved oxygen concentration (DO), pH and hardness of test solution: The water temperature values measured during the exposure ranged from 19.9 to 20.5 ⁰C, and they were within 20 ± 1 ⁰C. The measured DO values ranged from 8.4 to 8.9 mg/L, and they were above 60 % of air saturation value (The saturation values at 19 to 21 ⁰C: 9.01 to 8.68 mg/L, reference cited: JIS K 0102). The measured pH ranged from 7.4 to 7.9, and they met the requirement that pH should be within 6 - 9 and should not vary by more than 1.5 units. The measured values of hardness ranged from 52.0 to 62.0 mg CaCO3/L, and they were within 10 – 250 mgCaCO3/L. Thereby, it was thought that water temperature, DO, pH and hardness were within the range of suitable condition as the environment for the daphnid test.
- Test material concentration in test solution: The measured concentrations of the test material in the test solutions ranged from 95.0 to 111 % of the nominal at the preparation, and from 45.7 to 105 % of the nominal before renewal. Almost all values measured before renewal were above 80 % of the nominal, although only 2 measured values in 0.320 mg/L level were less than 80 % of the nominal, i.e. 77.4 % and 45.7 %. The reason for these lower recoveries is unclear, however, it seems that the loss of the test material may be based on the occurrence of the condition where it is easy to volatile at the time of handling the test solution, considering the physical-chemical property of the test material, the degree of accuracy of the chemical analysis, the closed condition of the vessel and the frequency of occurrence of decline of the test material concentration. Therefore, it is adequate to deem that the test material concentrations in test solution were close to the nominal concentration, and thus the test results was estimated using the nominal concentration.
Reported statistics and error estimates:
EC 50 value was estimated using binomial methods.

The 14 and 21 day EC50 values were 0.0205 and 0.230 mg/L The reason for the increased EC50 after 21 days is unclear, but it is thought that hormesis may be involved.

Table 2: Adult daphnid mortality and number of juveniles at Day 21 during the Definitive Test

Assessment at Day 21

Nominal concentration of the test material (mg/L)

0

0.02

0.04

0.08

0.16

0.32

Mortality of adults (%)

20

30

15

10

5a

20

Mean number of offspring per surviving parent

104

100

112

130

136

0.1

aaccidental death due to handling

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this 21-day semi-static reproduction study with the cladoceran, Daphnia magna, the NOEC was 0.16 mg/L.
Executive summary:

The long term toxicity of the test material to Daphnia magna was determined in accordance with the standardised guidelines OECD 211 and JMAFF 2-7-2-2, under GLP conditions.

JuvenileDaphnia magnawere exposed to a geometric series of five concentrations and a negative (dechlorinated tap water) control. Nominal test concentrations of 0.02, 0.04, 0.08, 0.16 and 0.32 mg/L were selected. Test solutions were renewed on a daily basis and the daphnids were transferred from the “old” to the “new” solutions. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at three intervals during the test. The concentrations of the old and new solutions were measured.  

Mortality of parent daphnids was observed on a daily basis. Size and appearance, swimming behaviour, determination of sex, presence of eggs in the brood pouch, ephippia and aborted broods were also recorded daily.From the onset of offspring production, the number of juveniles was counted on a daily basis and their condition was noted. The offspring were discarded after counting.

The cumulative mortality of the parent daphnids in the control, 0.0400, 0.0800, 0. 160 and 0.320 mg/L groups were 20, 30, 15, 10, 5 and 20 % respectively. Mortalities were not thought to be test material related. The mean number of live offspring per parent in the control group was 104. The mean cumulative numbers of live offspring in the 0.0800-0.160 mg/L test groups was between 100 and 136. In the 0.320 mg/L group the mean cumulative number of offspring was 0.1 and an adverse effect was obviously recognised. Instances of light body colour, growth inhibition and immobilisation were seen in the control, 0.0200, 0.0400 and 0.0800 mg/L groups. No abnormal appearances were observed in the 0.160 mg/L group. In the 0.320 mg/L group more parent daphnids showed light body colour and change to white colour of eggs in the brood pouch after 14 days, lethargic condition after 20 days and attachment of chlorella to body and immobilisation after 21 days. The time to first brood release or time to hatch was 8 days in all exposure groups except 0.320 mg/L where the mean value was 11.3 days (range 9-15 days).

The validity criteria for adult control mortality (not greater than 20 %) and mean number live offspring per surviving adult was greater than 60. The test was therefore considered valid.

 Under the conditions of this 21-day semi-static reproduction study with the cladoceran, Daphnia magna, the NOEC was 0.16 mg/L.

Description of key information

Under the conditions of this 21-day semi-static reproduction study with the cladoceran, Daphnia magna, the NOEC was 0.16 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.16 mg/L

Additional information

The long term toxicity of the test material to Daphnia magna was determined in accordance with the standardised guidelines OECD 211 and JMAFF 2-7-2-2, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

JuvenileDaphnia magnawere exposed to a geometric series of five concentrations and a negative (dechlorinated tap water) control. Nominal test concentrations of 0.02, 0.04, 0.08, 0.16 and 0.32 mg/L were selected. Test solutions were renewed on a daily basis and the daphnids were transferred from the “old” to the “new” solutions. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at three intervals during the test. The concentrations of the old and new solutions were measured.  

Mortality of parent daphnids was observed on a daily basis. Size and appearance, swimming behaviour, determination of sex, presence of eggs in the brood pouch, ephippia and aborted broods were also recorded daily.From the onset of offspring production, the number of juveniles was counted on a daily basis and their condition was noted. The offspring were discarded after counting.

The cumulative mortality of the parent daphnids in the control, 0.0400, 0.0800, 0. 160 and 0.320 mg/L groups were 20, 30, 15, 10, 5 and 20 % respectively. Mortalities were not thought to be test material related. The mean number of live offspring per parent in the control group was 104. The mean cumulative numbers of live offspring in the 0.0800-0.160 mg/L test groups was between 100 and 136. In the 0.320 mg/L group the mean cumulative number of offspring was 0.1 and an adverse effect was obviously recognised. Instances of light body colour, growth inhibition and immobilisation were seen in the control, 0.0200, 0.0400 and 0.0800 mg/L groups. No abnormal appearances were observed in the 0.160 mg/L group. In the 0.320 mg/L group more parent daphnids showed light body colour and change to white colour of eggs in the brood pouch after 14 days, lethargic condition after 20 days and attachment of chlorella to body and immobilisation after 21 days. The time to first brood release or time to hatch was 8 days in all exposure groups except 0.320 mg/L where the mean value was 11.3 days (range 9-15 days).

The validity criteria for adult control mortality (not greater than 20 %) and mean number live offspring per surviving adult was greater than 60. The test was therefore considered valid.

 

Under the conditions of this 21-day semi-static reproduction study with the cladoceran, Daphnia magna, the NOEC was 0.16 mg/L.