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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
vehicle was used
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: GG01
- Purity: 99.9% (GC method)
Analytical monitoring:
yes
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A respective amount of test substance and vehicle was added to synthetic freshwater in order to a treatment concnetration of 2 mg/L and a vehicle concentration of 100 mg/L.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamid, HCO-40
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 mg/L
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: National Institute for Environmental Studies
- Age of parental stock (mean and range, SD): < 24h
- Feeding during test: none

ACCLIMATION
- Acclimation period: Nov17th,1999 – Dec13th,1999. The death rate of parents 2 weeks before exposure was 0% and no occurrence of resting egg and male were recognized.
- Acclimation conditions (same as test or not): same
- Type and amount of food: Chlorella vulgaris
- Feeding frequency: 0.2 mg C /Daphnia / day
- Health during acclimation (any mortality observed): The death rate of parents 2 weeks before exposure was 0% and no occurrence of resting egg and male were recognize
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
not specified
Test temperature:
20 ± 1 °C
pH:
not specified
Dissolved oxygen:
not specified
Nominal and measured concentrations:
Nominal: 2 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Vessel with 100 mL test solution
- Type (delete if not applicable): covered water surface with teflon sheet
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic freshwater
- Intervals of water quality measurement: The pH was measured at the beginning of test and around the timing of water exchange every 24 hours.

OTHER TEST CONDITIONS
- Photoperiod: 16h light/ 8h dark
- Light intensity: room light

EFFECT PARAMETERS MEASURED: Immobilisation was recorded after 24 and 48h exposure time.
Reference substance (positive control):
not specified
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Validity criteria fulfilled:
yes
Conclusions:
The test substance is not acutely toxic to aquatic invertebrates within its limit of solubility in water.
Executive summary:

One acute toxicity study was performed to assess the acute toxicity of dicyclohexyl phthalate to the aquatic invertebrate Daphnia magna. The study was conducted under GLP and according to OECD guideline 202. The study was performed in a closed static system. Daphnids were exposed to the test substance at a limit test concentration of 2 mg/L using a vehicle to fascilitate exposure. Test substance analytics confirmed the nominal concentrations. The 48h-EC50 is determined to be > 2 mg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
vehicle was used
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: KN53200656
- Purity: 100% UVCB (isomers)
Analytical monitoring:
yes
Details on sampling:
The concentration of the saturated solution after 48 hours of stirring was measured. The measured test item concentration was close to or above the determined water solubility of the test item. At the start of the exposure and at the renewal (0 and 24 hours), samples were taken after preparation of the saturated solution and analyzed. At the renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken from the test vessels. The procedure for preparation of the samples is described below.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal loading of 100 mg/L of the test item was tested in a limit test. The limit loading level was selected based on the results of a non-GLP preliminary range finding test.
Saturated solutions with a nominal loading of 100 mg/L of the test item (corresponding to 93.5 µL/L) were prepared one, two and three days prior to the start of the exposure (at days -3, -2 and -1) as follows: Before the test item was taken from the test item container, the test item was checked for homogeneity. 200 mg of the test item (= 187 µL) were applied on a glass slide which was carefully placed on the bottom of a glass flask without disrupting the test item from the slide. The glass flask was carefully filled with 2000 mL of the dilution water. A slow stirring procedure on a magnetic stirrer was applied. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. Gentle stirring (to avoid formation of an emulsion) was carried out for 48 hours with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for 1 hour for separation of undissolved test item. Thereafter, the saturated solution was removed by siphoning of the homogenous liquid phase from the approximate center of the water body. The saturated solution was checked each time via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion) and was found negative. Undissolved test item was visually not present during the test. The solution prepared on day -3 was used for the coating phase, the solution prepared on day -2 was used at the start of the exposure phase and the solution prepared on day -1 was used for the renewal of the test solutions after 24 hours.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: clone 5
- Source: inhouse culture (originally obtained from Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), Berlin, Germany)
- Age of parental stock (mean and range, SD): < 24h
- Feeding during test: none
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
160 to 180 mg/L as CaCO3
Test temperature:
18 - 22 °C
pH:
7.21 - 7.90
Dissolved oxygen:
7.02 - 9.00 mg/L
Nominal and measured concentrations:
Nominal: 100 mg/L as loading rate
Measured (geom. mean): 64 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL glass beakers containing 20 mL test solution
- Renewal rate of test solution (frequency/flow rate): every 24h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- A coating phase of 24 hours was carried out for saturation of the glass walls of the test vessels with the test item in order to prevent adsorption of the test item. For this purpose, the coating solutions (the saturated solution and the control solution) were filled into the test vessels 24 hours before insertion of the daphnids. The test vessels with the coating solutions were stored for 24 hours under test conditions. Thereafter, the test vessels were emptied and refilled with freshly prepared solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4
- Culture medium different from test medium: no
- Intervals of water quality measurement: Prior to the start of the exposure (0 hours) and the water renewal (24 hours), the water quality parameters (i.e. pH-value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured. The incubator temperature (measurement in air with a thermo-hygrograph) was recorded throughout the period of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 1500 lx

EFFECT PARAMETERS MEASURED: Immobilisation was recorded after 24 and 48h exposure time.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 64 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
No immobilisation was observed in the treatment and the control, respectively.
The saturated solution was checked each time via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion) and was found negative. The saturated solution was visually clear throughout the exposure period.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 24h-EC50 = 2.1 mg/L, which is within the valid range of 0.6 - 2.1 mg/L as given in the OECD guideline 202.

The measured concentrations in the fresh media were 53.7 µg/L at the start of the exposure (0 hours) and 111 µg/L at the renewal of the test solution (24 hours) and thus both above the water solubility limit. In the old media at renewal and at the end of the test (24 and 48 hours), the measured concentrations were 31.0 µg/L and 90.9 µg/L indicating a slight decrease. The geometric mean measured test item concentration was 64.0 µg/L.

Validity criteria fulfilled:
yes
Conclusions:
The test substance is not harmful to aquatic invertebrates at short-term exposure.

Description of key information

EL50(48h) > 100 mg/L (nominal, loadimg rate) for immobilisation of Daphnia magna (OECD 202)

Key value for chemical safety assessment

Additional information

One study investigating the short-term toxicity of Bis(methylcyclohexyl) phthalate (CAS 27987-25-3) to aquatic invertebrates is available. The study was performed according to GLP and OECD guideline 202 using Daphnia magna as test organism (Scheerbaum 2018). A 100 mg/L loading rate was tested in a limit test under semi-static conditions. Test substance analytics resulted in a geometric mean value of 64 mg/L. No immobilidsation was observed in the treatment and the control throughout the test. Thus, the 48h-EL50 value is determined to be > 100 mg/L based on nominal concentrations.

In addition, one study with the source substance used to cover the algal toxicity endpoint is available. The study was performed by the Japanese environmental agency under GLP according to OECD guideline 202 using Daphnia magna as test organism (MOE 2000). Dimethylformamid, HCO-40 were used to fascilitate the solubility of the substance in the test solution. A limit test concentration of 2 mg/L was tested. The nominal concentration was varified analytically. The 48h-EC50 value is deternined to be > 2 mg/L. Thus, the substance is not harmful to aquatic invertebrates up to its limit of solubility in water.

The identical results obtained for the target and the source substance support the suitability of using data from the source substance to cover the algal toxicity endpoint of the target substance.

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