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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Dec 2008 to 11 March 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to GLP and internationally accepted test guidelines.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
NON-ADJUSTED MEDIA
- Concentrations: 4.38, 10.7, 19.1, 45.5, 93.9 mg/L
- Sample storage conditions before analysis: Not applicable - samples analysed without storage (note that contingency samples were stored frozen, but not used).

pH-ADJUSTED MEDIA
- Concentrations: 4.35, 9.84, 18.6, 43.9, 95.4 mg/L
- Sample storage conditions before analysis: Not applicable - samples analysed without storage (note that contingency samples were stored frozen, but not used).
Vehicle:
no
Details on test solutions:
Two aqueous stock solutions were prepared by the direct addition of the test substance (107 μL, corrected for specific gravity) in dilution medium (1 L) in volumetric flasks. The contents of the flasks were shaken vigorously and serially diluted to provide two sets of test media, each at 4.27, 9.39, 20.7 and 45.5 mg/L (1 L final volume).
The first set of media was tested without adjustment. For the second set, the pH of the medium at each concentration was checked and then adjusted to within the pH range of 7.4 to 8.2 using 0.1M hydrochloric acid.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: daphnia magna (Straus)
- Age at study initiation (mean and range, SD): first instar (less than 24 hours)
- Method of breeding: parthenogenesis
- Feeding during test
- Food type: unicellular green algae
- Amount: 0.1 to 0.2 mg carbon per daphnid per day

ACCLIMATION
- Acclimation period: not specified
- Acclimation conditions (same as test or not): yes
- Type and amount of food: 0.1 to 0.2 mg carbon
- Feeding frequency: once per day
- Health during acclimation (any mortality observed): not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20±2°C
pH:
Non-adjusted media: 7.66 (control) - 10.12 (100 mg/L nominal dose level)
pH-adjusted media: 7.62 - 7.98
Dissolved oxygen:
101 - 109% Air Saturation Value
Nominal and measured concentrations:
Nominal: 4.27, 9.39, 20.7, 45.5, 100 mg/L
Measured, unadjusted media: 4.38, 10.7, 19.1, 45.5, 93.9 mg/L
Measured, pH adjusted media: 4.35, 9.84, 18.6, 43.9, 95.4 mg/L
Details on test conditions:
TEST SYSTEM
- No. of organisms per vessel:5
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates):5
-Biomass loading: 5 organisms per 100ml

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test organisms were maintained and the tests conducted in Elendt M4 medium. The medium was prepared in deionised, reverse osmosis water.

OTHER TEST CONDITIONS
- Adjustment of pH:The first set of media was tested without adjustment. For the second set, the pH of the
medium at each concentration was checked and then adjusted to within the pH range of 7.4 to
8.2 using 0.1M hydrochloric acid.
- Photoperiod:16 hours light:8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
immobility at 24 and 48 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations: 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: At 100 mg/L, pH was >10. A second rangefinding test at 100 mg/L was conducted with non-adjusted and pH-adjusted media. After 48 hours, all Daphnia in both media types were immobile. Test concentrations were 4.27, 9.39, 20.7 and 45.5 mg/L.
Duration:
48 h
Dose descriptor:
EC50
Remarks:
(no pH adjustment)
Effect conc.:
33.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% Confidence limits: 27.0 and 39.6 mg/L.
Duration:
48 h
Dose descriptor:
EC50
Remarks:
(pH adjusted media)
Effect conc.:
65.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 45.5 and 93.9 mg/L.
Duration:
48 h
Dose descriptor:
NOEC
Remarks:
(no pH-adjustment)
Effect conc.:
19.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Remarks:
(pH adjusted media)
Effect conc.:
43.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The results obtained suggest that the increase in pH caused by the presence of 1,3-bis(aminomethyl)cyclohexane was partially responsible for the higher level of toxicity to Daphnia magna observed in the non-adjusted media when compared to the pH-adjusted media 65.4

Parameter (mg/L)

Non-adjusted

pH-adjusted

24 hour EC50

68.6

75.0

95% confidence limits

60.1 & 93.9

68.7 & 93.9

48 hour EC50

33.1

65.4

95% confidence limits

27.0 & 39.6

45.5 & 93.9

Lowest concentration where 100% immobility occurred

93.9

95.4

“No observed effect concentration”

19.1

43.9

In non-adjusted media, the intended exposure levels of 1,3-bis(aminomethyl)cyclohexane were adequately achieved at all concentrations (between 90 and 110% of their nominal values) and maintained during the test (between 103 and 113% of their starting values). The overall mean measured levels were 4.38, 10.7, 19.1, 45.5 and 93.9 mg/L and these values have been used in the calculation of test results.

In pH-adjusted media, the intended exposure levels of 1,3-bis(aminomethyl)cyclohexane were adequately achieved at all concentrations (between 86 and 102% of their nominal values) and maintained during the test (between 106 and 113% of their starting values). The overall mean measured levels were 4.35, 9.84, 18.6, 43.9 and 95.4 mg/L and these values have been used in the calculation of test results.

Validity criteria fulfilled:
yes
Conclusions:
The 48-hour EC50 of 1,3-bis(aminomethyl)cyclohexane for the immobilisation of Daphnia magna was 33.1 mg/L (95% confidence limits of 27.0 and 39.6 mg/L) when tested without pH adjustment and 65.4 mg/L (95% confidence limits of 45.5 and 93.9 mg/L) when the pH of the media was adjusted to within the recommended range for this type of test.
The ‘no-observed effect concentration’ of 1,3-bis(aminomethyl)cyclohexane with Daphnia magna was 19.1 mg/L in non-adjusted media and 43.9 mg/L where the pH of the media was adjusted.
These results suggest that the pH of the test media, increased by the presence of 1,3- bis(aminomethyl)cyclohexane, was partially responsible for the toxicity observed in nonadjusted media.
Executive summary:

The acute toxicity to daphnia of the test material 1,3-Bis(aminomethyl)cyclohexane (1,3-BAC) was assessed in a study performed according to OECD test guideline 202 and EC guideline C2 and in compliance with GLP.

Test groups of Daphnia Magna Straus were exposed over 48 hours to the test material at concentrations 4.27, 9.39, 20.7, and 45.5, and 100 mg/L, and the daphnia were exposed both to pH adjusted media (adjusted to account for the elevated pH found in solutions of the test material) as well as unadjusted media.

In non-adjusted media, the 48-hour EC50was calculated to be 33.1 mg/L and the No Observed Effect Concentration (NOEC) was 19.1 mg/L. In pH adjusted media the EC50was 65.4 mg/L and the NOEC was 43.9 mg/L. These results suggested that the increased pH of test solutions containing 1,3-BAC had contributed to the toxicity.

Description of key information

Daphnia magna, 48-hours, test medium pH not adjusted:  LC50 = 33.1 mg/L (95% CI of 27.0 and 39.6 mg/L)(Values based on measured concentrations).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
33.1 mg/L

Additional information

The acute toxicity to daphnia of the test material 1,3-Bis(aminomethyl)cyclohexane (1,3-BAC) was assessed in a study performed according to OECD test guideline 202 and EC guideline C2 and in compliance with GLP.

Test groups of Daphnia Magna Straus were exposed over 48 hours to the test material at concentrations 4.27, 9.39, 20.7, and 45.5, and 100 mg/L, and the daphnia were exposed both to pH adjusted media (adjusted to account for the elevated pH found in solutions of the test material) as well as unadjusted media.

In non-adjusted media, the 48-hour EC50 was calculated to be 33.1 mg/L and the No Observed Effect Concentration (NOEC) was 19.1 mg/L. In pH adjusted media the EC50 was 65.4 mg/L and the NOEC was 43.9 mg/L. These results suggested that the increased pH of test solutions containing 1,3-BAC had contributed to the toxicity.