Registration Dossier

Diss Factsheets

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
1994-10-26 to 1994-10-28
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study with no acceptable restrictions: an emulsion was prepared with a surfactant to reach concentrations well above sulubility limit. Varying concentration of surfactant in the different test substance concentrations make impossible to identify concentration/effect relying only to the test substance
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
no details mentioned
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of stock solution: 1.03 g test substance was mixed with 1.03 g Vehicle and made up to 1 L with deionised water.
- Controls: yes, deionised water and vehicle control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Castor oil, ethoxylated (40 EO)
- Concentration of vehicle in test medium (stock solution and final test solution): The concentration of vehicle is the same as the test substance in every test solution and in the stock solution. The vehicle control contains 2.0 mg/L castor oil, ethoxylated
- Evidence of undissolved material (e.g. precipitate, surface film, etc): nothing mentioned
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain: Daphnia magna Straus, clone 5
- Source: laboratory breed
- Age at study initiation: not older than 24 hours
- Method of breeding: The females were maintained in M4 medium in 1 l beakers. A reddish dark-brown coloration of the animals indicated that they were healthy. At two to three day intervals, the exuviae were siphoned off and the water was changed. In the course of this, the offspring was removed from the breeding vessels. At regular intervals of approx. 4 weeks, offspring was isolated from the vessels. They served as a basis for further breeding.
- Feeding during test: none



ACCLIMATION
- Acclimation period: Females were seperated from the offspring 24 hours before the start of the test. The offspring produced overnight were transferred into test water and were used for the test.
- Type and amount of food: Scenedesmus subspicatus, the amount of food added is such that it is consumed by the animals
- Feeding frequency: daily
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
none
Hardness:
2.5 mmol
Test temperature:
20 °C ± 1 °C
pH:
7.9 - 8.0
Dissolved oxygen:
8.3 - 8.4 mg/L
Salinity:
CaCl2 x 2 H2O: 294 mg/L; MgSO4 x 7 H2O: 123 mg/L; NaHCO3: 63 mg/L; KCl: 5.5 mg/L
Ca/Mg ratio: 4:1
Na/K ratio: 10:1
Nominal and measured concentrations:
0.13 / 0.25 / 0.50 / 1.0 / 2.0 mg/L
(The geometric mean after 24 and 48 h of the analytically determined test solutions differed < 20 % from the nominal concentrations, therefore the nominal concentrations were used for evaluation.
Details on test conditions:
TEST SYSTEM
- Test vessel: Round-bottom test tubes
- Material, size, headspace, fill volume: glass, calibrated to 10 mL
- Aeration: none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic fresh water (CaCl2 x 2 H2O: 294 mg/L; MgSO4 x 7 H2O: 123 mg/L; NaHCO3: 63 mg/L; KCl: 5.5 mg/L)
- Ca/mg ratio: 4:1
- Conductivity: not mentioned
- Culture medium different from test medium: yes
- Intervals of water quality measurement: not mentioned


OTHER TEST CONDITIONS
- Adjustment of pH: not adjusted
- Photoperiod: not applicable
- Light intensity: no light during test


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation after 24 and 48 hours; O2 concentration, pH at the end of the test


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not mentioned
- Range finding study: not mentioned
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 0.77 - 2.1 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Details on results:
- Behavioural abnormalities: nothing mentioned
- Observations on body length and weight: not applicable
- Other biological observations: nothing mentioned
- Mortality of control: not examined
- Other adverse effects control: 0% (immobilisation)
- Abnormal responses: nonr
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: nothing mentioned
- Effect concentrations exceeding solubility of substance in test medium: none
Results with reference substance (positive control):
- Results with reference substance valid? yes, historical control (not tested in parallel to the test substance)
- Concentrations: 1.0 / 2.0 mg/L potassium dichromate
- Results: 45 % immobilisation at 1.0 mg/L potassium dichromate after 24 h; 100 % immobilisation at 2.0 mg/L potassium dichromate after 24 h
Reported statistics and error estimates:
no statistics performed, the EC50 values were calculated graphically (dose-response relationship).

Table #1: Effect of Dibenzylbenzene, ar-methyl derivative on immobilisation of Daphnia magna

  concentration [mg/L]        24 hour values        48 hour values
      number  %  number   
   mobile  immobile  immobile  mobile  immobile immobile 
 0 20   0  0  20  0  0
vehicle control  20  0  0  20  0  0
 0.13  20  0  0  20  0  0
 0.25  20  0  0  20  0  0
 0.50  20  0  0  15  5  25
 1.0  20  0  0  15  5  25
 2.0  20  0  0  5  15  75
Validity criteria fulfilled:
yes
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
1990-08-29 to 1990-08-31
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study with no acceptable restrictions: an emulsion was prepared with a surfactant to reach concentrations well above sulubility limit. Varying concentration of surfactant in the different test substance concentrations make impossible to identify concentration/effect relying only to the test substance
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
The test period in this study was 43 hours. Despite this deviation from the guideline the test was considered to be valid from the author of the study.
GLP compliance:
yes
Specific details on test material used for the study:
The purity of the material used for the test was lower than the purity of the actual material. It contained ca. 26% of isomers of dimethyl tribenzenes. However, based on comparable basic structure of the impurities and the assumption that any toxic effects would increase with higher molecular weight and lipophilicity, we regard that the studies conducted with the lower purity material being "worst case" and can be used for the evaluation of the actual product.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control; vehicle control; 0.06; 0.13; 0.25; 0.5; 1.0 mg/L (after 0 h and 43 h post exposure) and spiked water samples of 0.10; 0.30; 1.00 mg/L Marlotherm S
- Sampling method: not mentioned
- Sample storage conditions before analysis: not mentioned
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 13,54 g of vehicle (Marlowet R 40) were weighed into a 100 mL glass vessel, 499,7 mg Marlotherm S were added. This mixture was stirred and 35.96 g reconstituted water were added. After 15 minutes 0.5 mL of the stock solution was added to 5000 mL reconstituted water. Concentration of diluted stock solution: 1 mg/L. Storage conditions for diluted stock solution: light, 22 ± 1 °C, 300 min.
- Controls: yes, reconstituted water and vehicle control, reference control (not tested in parallel, historical control)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Ricinoleicacid ethoxylate
- Concentration of vehicle in test medium (stock solution and final test solution): approx. 200 mL/L in the stock solution; 0.02 mL/L in the diluted stock solution; in the final test solutions ranging from 0.003125 to 0.025 mL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): nothing mentioned
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain: Daphnia magna Straus
- Source: Hoechst AG, Dr. Voelskow, Frankfurt a.M., Germany
- Age at study initiation (mean and range, SD): 6 - 19 h, no further details mentioned
- Weight at study initiation (mean and range, SD): not mentioned
- Length at study initiation (length definition, mean, range and SD): not mentioned
- Method of breeding: Daphnids were held in batches of 20 in reconstituted water (micro filtrated, "Millipore" MGCGL 10503, additives: 2 µg/L Na-Selenate and 2 µg/L Vitamine B12) in vessels made of glass (size: 3.5 L; height: 10 cm; diameter: 22.5 cm). The amount of reconstituted water in the stock vessels was 2 L , the depth of water was 5.5 cm. The water was renewed batchwise at least twice per week. Temperature of the room: 22 ± 1 °C; photoperiod: light/datrk (16/8 h); quantum flow density: approx. 20 µE/s x m²; aeration: no. The daphnids were fed at least twice per week with a mixture of algae (Scenedesmus subspicatus and Chlorella vulgaris) and organic additive (Aquarium fish food: TetraSpecialMix). Adult and young daphnids were separated at öeast twice per week. The quality criteria of the stock culture of daphnids were given as: 1. Reproduction rate per adult (average value): approx. 10 daphnids per day (determined Aug. 29, 1990); 2. Microscopical examination of health contions: normal (performed: Aug. 29, 1990).
- Feeding during test: none


ACCLIMATION
- Acclimation period: 24 h before the test was started adult daphnids were collected and cultured in reconstituted water for 18 h. Then the newborn daphnids were collected and held for 6 h.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: not mentioned
- Health during acclimation (any mortality observed): no mortality mentioned
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
43 h
Post exposure observation period:
not performed
Hardness:
213.6 mg/L CaCO3
Test temperature:
21.1 - 21.7 °C
pH:
7.6 - 8.0
Dissolved oxygen:
101 - 114 %; 8.7 - 9.8 mg/L
Salinity:
294 mg/L CaCl2 x 2 H2O
123.75 mg/L MgSO4 x 7 H2O
64.75 mg/L NaHCO3
5.75 mg/L KCl
Nominal and measured concentrations:
control / vehicle control / 0.06 / 0.13 / 0.25 / 0.50 / 1.00 mg/L (nominal and measured)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed (covered by glass lids)
- Material, size, headspace, fill volume: material: glass; size: 300 mL (height: 5 cm; diameter: 9 cm); headspace: 1 cm; fill volume: 250 mL
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Four solutions (A, B, C, D) were prepared in deionized water:
A) 11.76 g/L CaCl2 x 2 H2O
B) 4.93 g/L MgSO4 x 7 H2O
C) 2.59 g/L NaHCO3
D) 0.23 g/L KCl
25 mL of each of the four solutions were mixed and made up to 1 L with deionized water and aerated for several hours (= reconstitued water).
- Culture medium different from test medium: yes
- Intervals of water quality measurement: Chemical characteristics of the reconstituted water were determined after preparation.


OTHER TEST CONDITIONS
- Adjustment of pH: not adjusted
- Photoperiod: light/dark (16/8 h)
- Light intensity: not mentioned


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilization after 24 and 43 hours; O2 content, pH and temperature: 14 measurements were made during test, intervals not mentioned


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not stated
- Range finding study: not performed, toxicity data provided by the sponsor were used to set the concentration levels for the main test
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
43 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
43 h
Dose descriptor:
EC0
Effect conc.:
>= 1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: not mentioned
- Observations on body length and weight: not determined
- Other biological observations: none
- Mortality of control: not applicable
- Other adverse effects control:
Immobilization: 0 %
Trapping of daphnids at the water surface: 0 %
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Mortality: not mentioned
- EC50/LC50: EC50 (24 h) = 1.31 mg/L
- Other: not tested in parallel, test performed 1989-08-01 until 1989-08-03
Reported statistics and error estimates:
no statistics performed
Validity criteria fulfilled:
yes
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
1990-02-06 to 1990-03-22
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study with no acceptable restrictions: an emulsion was prepared with a surfactant to reach concentrations well above sulubility limit. Varying concentration of surfactant in the different test substance concentrations make impossible to identify concentration/effect relying only to the test substance
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes
Specific details on test material used for the study:
The purity of the material used for the test was lower than the purity of the actual material. It contained ca. 26% of isomers of dimethyl tribenzenes. However, based on comparable basic structure of the impurities and the assumption that any toxic effects would increase with higher molecular weight and lipophilicity, we regard that the studies conducted with the lower purity material being "worst case" and can be used for the evaluation of the actual product.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: same as in test
- Sampling method: not mentioned
- Sample storage conditions before analysis: not mentioned
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of a stock solution of 1000 mg/L test substance
- Controls: yes, blank and vehicle control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Ricinoleicacid ethoxylate
- Concentration of vehicle in test medium (stock solution and final test solution): 1000 mg/L in the stock solution; final concentrations same as test substance
- Evidence of undissolved material (e.g. precipitate, surface film, etc): nothing mentioned
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain: Daphnia magna Straus, clone 5
- Source: laboratory breed
- Age at study initiation: not older than 24 hours
- Method of breeding: The females were maintained in dechlorinated tap water in 1 L beakers. The daphnids were fed with a mixture of algae (Chlorella vulgaris) and aqueous extract of Trouvit trout food on a daily basis, the amount of food added is such that it is consumed by the animals. A reddish dark-brown coloration of the animals indicated that they were healthy. At two to three day intervals, the exuviae were siphoned off and the water was changed. In the course of this, the offspring was removed from the breeding vessels. At regular intervals of 4 - 5 weeks, offspring was isolated from the vessels. They served as a basis for further breeding.
- Feeding during test: none



ACCLIMATION
- Acclimation period: Females were seperated from the offspring 24 hours before the start of the test. The offspring produced overnight were transferred into test water and were used for the test.
- Type and amount of food: not mentioned
- Feeding frequency: not mentioned
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
none
Hardness:
2.5 mmol
Test temperature:
20 °C ± 10 °C
pH:
7.7 - 8.4
Dissolved oxygen:
8.1 - 8.7 mg/L
Salinity:
CaCl2 x 2 H2O: 294 mg/L; MgSO4 x 7 H2O: 123 mg/L; NaHCO3: 63 mg/L; KCl: 5.5 mg/L
Ca/Mg ratio: 4:1
Na/K ratio: 10:1
Nominal and measured concentrations:
Nominal: 0.06 / 0.10 / 0.17 / 0.31 / 0.55 / 1.00 mg/L
Measured: after 0 hours: 0.06 / 0.11 / 0.17 / 0.30 / 0.52 / 0.95 mg/L
after 48 hours: 0.043 / 0.088 / 0.16 / 0.28 / 0.50 / 0.96 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Round-bottom test tubes
- Material, size, headspace, fill volume: glass, calibrated to 10 mL
- Aeration: none
- No. of organisms per vessel: 5 (in one concentration (0.10 mg/L) only 4 daphnids in one replicate)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): blank: 4; vehicle control: 3


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic fresh water (CaCl2 x 2 H2O: 294 mg/L; MgSO4 x 7 H2O: 123 mg/L; NaHCO3: 65 mg/L; KCl: 6 mg/L)
- Ca/mg ratio: 4:1
- Conductivity: not mentioned
- Culture medium different from test medium: yes
- Intervals of water quality measurement: not mentioned


OTHER TEST CONDITIONS
- Adjustment of pH: not adjusted
- Photoperiod: not applicable
- Light intensity: no light during test


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation after 24 and 48 hours; O2 concentration, pH at the end of the test


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not mentioned
- Range finding study: not mentioned
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 0.4 - 0.9 mg/L 95% CL
Key result
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
ca. 0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: none
- Observations on body length and weight: not determined
- Other biological observations: The animals were adhered more or less to the surface of the test solutions
- Mortality of control: not applicable
- Other adverse effects control: blank 10% immobilised daphnids; vehicle control 0% immobilised daphnids
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: nothing mentioned
Results with reference substance (positive control):
- Results with reference substance valid? yes, historical control (not tested in parallel to the test substance)
- Concentrations: 0.9 / 1.9 mg/L potassium dichromate
- Results: 35 % immobilised daphnids at 0.9 mg/L, 60 % at 1.9 mg/L potassium dichromate after 24 h
Reported statistics and error estimates:
Calculation of EC50 with probit analysis according to Cavalli-Sforza (1972)

Table #1: Effect of Marlotherm S on immobilisation of Daphnia magna

  concentration [mg/L]        24 hour values        48 hour values
      number  %  number   
   mobile  immobile  immobile  mobile  immobile immobile 
 0 (control)  18  2  10  18  2  10
 0 (vehicle control)  20  0  0  20  0  0
 0.06  20  0  0  19  1  5
 0.10*  18  1  5.3  18  1  5.3
 0.17  18  2  10  15  5  25
 0.31  17 3  15  15  5  25
 0.55  15  5  25  9  11  55
 1.00  13  7  35  8  12  60

* only 19 daphnids used

Validity criteria fulfilled:
yes
Conclusions:
It has to be taken into consideration that a part of the individuals got stuck at the surface film of the test vessels. This could not be eliminated even after several repetitions of the test, so that the repoducibility of the results were not completely guaranteed under such conditions.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Principles of method if other than guideline:
Paris Commission Guidelines Regarding Harmonisation of Procedures of Approval, Evaluation, and Testing of Offshore Chemicals and Drilling Muds (1990).
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Duplicate aliquots were removed from stock solutions at the commencement of each test and from the contents of each tank at the end of each test, for determination of test material concentration by analysis of total radioactivity.
Vehicle:
no
Details on test solutions:
For the range finding and definitive tests, stock suspensions were prepared by adding 1 litre of filtered seawater to 45.4 µg [14C]-DBT and subjecting the mixture to ultrasonication (20 min) followed by continuous stirring (18-24 h). The suspension was centrifuged (1000 r.p.m., 30 min) using a Jouan model C412 bench centrifuge and total radioactivity measured in the supernatant fraction which was used for testing.

For the definitive test, the supernatant fraction was diluted with filtered seawater to produce the following measured concentrations:

20.6, 9.0, 4.5, 2.3 and 1.1 µg equiv.l-', together with a control (0 µg equiv.l-')

Four tanks were prepared at each concentration, each containing 50 ml of test solution. Five Acartia were added to each tank.
Test organisms (species):
other aquatic crustacea: Acartia tonsa
Details on test organisms:
Acartia cultures were maintained in 10 litre glass tanks containing 10 µm filtered natural seawater. The seawater was replaced continuously using a flow-through system. Acartia cultures were fed on a diet of Rhodomonas sp and Skeletonema costatum. Cultures were fed continuously until the day of study commencement and were not fed during the test.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
no
Test temperature:
21 ± 0.5 °C measured
pH:
7.9-8.0 at test start and 8.1 at end
Dissolved oxygen:
> 96% saturation at start, > 91% at end
Salinity:
Filtered (10 µm) natural seawater was used for maintenance of cultures and as the test medium. The seawater was collected from a site on the East coast of Scotland and was free from known sources of pollution. The seawater typically has a salinity of 32 ± 2% and pH of 8.0 ±0.3.
Nominal and measured concentrations:
measured concentrations: 20.6, 9.0, 4.5, 2.3 and 1.1 µg equiv.l-', together with a control (0 µg equiv.l-')
Details on test conditions:
Culture and test tanks were maintained within the laboratory at a temperature of 20 ± 2°C. A light cycle of 16 h light and 8 h dark applied over the study period, illumination being provided by artificial daylight fluorescent tubes.
Reference substance (positive control):
yes
Remarks:
3.5 dichlorophenol
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
9 µg/L
Basis for effect:
mortality
Remarks on result:
other: ANOVA
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 20.6 µg/L
Basis for effect:
mortality
Details on results:
number of dead Acartia were recorded at 24 h and 48 h.
An Acartia was considered dead if no movement was observed within 10 s following gentle agitation of the test tanks.
Results with reference substance (positive control):
In accordance with UK MAFF guidelines (1993) a reference toxicant (3,5-Dichlorophenol at 1.0 mg/L) was tested during the definitive test. Four tanks containing reference toxicant and 4-5 Acartia were included in the test. The following mortalities were recorded:
Time Cumulative Number of
(h) Mortalities (%)
24 58
48 63
As the percentage mortality after 48 h exposure falls within the acceptance criteria of 20-80% mortalities as specified by UK MAFF, the test is considered to have been valid.

The maximum solubility achieved was 20.6 µg equiv/L. This decreased to 3.9 µg equiv/L alter 48 h also suggesting that the chemical became bound to the test tanks during the test. Similar results were found at the other treatment concentrations and no radioactivity was detected in control tanks throughout the test.

At the highest concentration tested, 15% and 40% mortalities were observed after 24 h and 48 h respectively. After 48 h, 10% mortalities were recorded in the control tanks, between 5% and 10% mortalities were recorded at initial concentrations of between 1.1 and 4.5 µg equiv/L and 15% mortalities at 9.0 µg equiv/L.

Validity criteria fulfilled:
yes
Remarks:
result on reference substance
Conclusions:
At 24 h, the No Observed Effect Concentration (NOEC) was at least 20.6 µg equiv/L, the optimum solubility. At 48 h, the NOEC was 9.0 µg equiv/L and the median lethal concentration (LC50) was greater than 20.6 µg equiv/L
Executive summary:

Acute toxicity study of Dibenzylbenzene, ar-methyl derivativeto wards marine zooplanction (Acartia tonsa) was carried out according to OSPAR Convention draft protocol (now ISO 14669). Several attemps were made for reaching stable concentrations. Maximum achieved concetration in sea water was 20.6 µg/L. Median lethal concentration LC50 was not reached for a exposure period of 48 h (lethality at this measured initial concentration was 40%). NOEC was 9 µg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Principles of method if other than guideline:
United Kingdom proposal to ISO/TC/147/SC5/WG2: Water quality - Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea)
GLP compliance:
yes
Remarks:
lab accredited by Norwegian Authority
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
For each test concentration a separate stock solution was prepared, by dissolving the test substance in acetone so that adding 100 µl of stock solution pr. litre of seawater would give the specified test concentration. The concentration of acetone in the controls was 100 µl/l
When preparing test solutions some test substance could visually be observed floating on the water surface at concentrations of 0,5 mg/1 and above, indicating that the solubility in seawater was exceeded.
Test organisms (species):
other aquatic crustacea: Acartia tonsa
Details on test organisms:
The test organisms were supplied as eggs, and were cultivated in accordance with the "Paris Commission - Ring Test Protocol" (VKI). Large copepodites and adults were used in the test.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
no
Hardness:
no data
Test temperature:
20 ± 1 °C
pH:
8.2 at test start and 8.3 at end
Dissolved oxygen:
6.6 mg/L
Salinity:
Natural seawater (salinity: 34 %o) from an unpolluted site in Byfjord was used as basic for the dilution water. The water was filtered through a GF/C filter prior to use.
Nominal and measured concentrations:
Nominal :
0,011 mg/I
0,027 mg/I
0,050 mg/I
0,10 mg/I
0,20 mg/I
0,49 mg/I
1,00 mg/I
1,99 mg/I
Details on test conditions:
The test was run in glass containers (28 ml rolled rim vials) each containing 25 ml of test solution and 5 animais, 4 replicates for each concentration. The vials were kept under dim continuous light (1500 - 2000 lux) at a temperature of 20 ± 1 °C. pH and oxygen measurements were performed as indicated on the data sheet.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.027 mg/L
Remarks on result:
other: ANOVA
Key result
Duration:
48 h
Dose descriptor:
other: LC90
Effect conc.:
0.62 mg/L
Remarks on result:
other: Probit
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
0.087 mg/L
Remarks on result:
other: Probit 95% CL: 0,054 - 0,132
Key result
Duration:
48 h
Dose descriptor:
LC10
Effect conc.:
0.012 mg/L
Remarks on result:
other: Probit
Details on results:
based on lethality
Results with reference substance (positive control):
LC50-48h = 18 mg/L
Validity criteria fulfilled:
yes
Conclusions:
LC50-48h = 0.087 mg/L based on nominal concentrations
Executive summary:

Acute toxicity towards marine copepods was assessed according to a draft ISO standard. Lethality is the end-point. There is uncertainty around reliability arises from the use of solvent and testing above solubility. No analysis of exposure concentration was carried out. The LC50 being in the range of solubility can be used as a supporting study.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to guideline, GLP, labelled test substance measured
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
1ml test solution is transferred by pipetting to LSC vials, exact quantity checked by weighting, 10 ml of Ultimagold Packard scintillation liquid is added, and counting is done without intermediate storage
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 14C-DBT is available in isopropanol solution. 16,5 µl (30 µg DBT) is introduced in 1L test medium and stirred vigorously during 48h, settled, then transferred in tests flasks
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): isopropanol
- Concentration of vehicle in test medium (stock solution and final test solution): 16,5 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: Daphnia magna
- Strain: "clone 5" (also known as "clone A")
- Source: IRCHA
- Age at study initiation (mean and range, SD): < 24h
- Method of breeding: in lab, in Elendt M7 medium, fed with Raphidocelis subcapitata
- Feeding during test: no

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
no
Hardness:
test medium according to ISO 6341
Ca+Mg ions = 2.5 mmol/L
Test temperature:
18-20°C
pH:
7.5 in limit test solution, 7.76 in blank medium
Dissolved oxygen:
7.7 mgO2/L in limit test solution, 8.3 in blank medium
Salinity:
test medium according to ISO 6341
Nominal and measured concentrations:
Limit (saturated) test initial measured concentration : 0.029 mg DBT/L
Limit (saturated) test final measured concentration : 0.027 mg DBT/L
Final/initial = 93%
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: penicillin type 120 ml, closed with sealed PTFE septa, fill volume : 120 mL (no headspace)
- 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: ultra-pure water
- Ca/mg ratio: 4
Components:
11.76 g CaCl2, 2H2O/L
4.93 g Mg SO4, 7H2O/L
2.59 g ofNaHCO3/L
0.23 g of KCl/L

- Light intensity: protected from light


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
mobility

TEST CONCENTRATIONS
Saturated solution (measured value = 0.029 - 0.027 mg/L)
- Justification for using less concentrations than requested by guideline: poor solubility: limit test at WSL
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 0.029 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: water solubility limit in test conditions measured through 14C
Key result
Duration:
48 h
Dose descriptor:
LC0
Effect conc.:
>= 0.029 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: water solubility limit in test conditions measured through 14C
Details on results:
QUALITY CRITERIA:
- Control mortality (OK if < 10%): OK
- Daphnia in control were not trapped at the surface of the water: no
- pH (OK if equals to initial pH ± 1): OK
- Dissolved oxygen (OK if > 2mg/L): OK
- Test substance concentration (OK if > 80% of initial concentration): OK
Validity criteria fulfilled:
yes
Remarks:
QUALITY CRITERIA: - Control mortality (OK if < 10%): OK - Daphnia in control were not trapped at the surface of the water: no - pH (OK if equals to initial pH ± 1): OK - Dissolved oxygen (OK if > 2mg/L): OK - Test substance concentration (OK if > 80% of i
Conclusions:
No immobilisation of daphnia was observed for exposure during 48 h at solubility limit in test medium. Therefore EC50 of Dibenzylbenzene, ar-methyl derivative at 48 h for Daphnia magna was determined to be > 0.029 mg/L, found to correspond to the solubility limit of the tested substance in the mineral medium used for this test.
Executive summary:

The acute toxicity (inhibition of mobility) of Dibenzylbenzene, ar-methyl derivative for Daphnia magna was assessed according to the method C2 of the European Directive 92/69/CEE (equivalent to OECD 202). Daphnia were exposed in a static test to a concentration of about 0,029 mg/l. The test was performed with 5 daphnia per vessel. The test was performed using closed flasks entirely filled with test solutions and closed with butyl rubber caps covered with PTFE. The test concentrations of Dibenzylbenzene, ar-methyl derivative were measured by Liquid scintillation counting.

No inhibition of mobility was observed after 24 or 48 hours at a tested concentration of about 0,029 mg/L of Dibenzylbenzene, ar-methyl derivative. This concentration is the solubility limit of Dibenzylbenzene, ar-methyl derivative in the mineral medium used for the test.

The method was applied with respect to its quality criteria :

Immobilisation in the control did not exceed 10 % at the end of the test ;

Concentration of dissolved oxygen in the test vessels remained above 2 mg/L at the end of the test and pH did not vary by more than 1 unit ;

The concentrations of the test substance have been maintained to within 80 % of the initial concentration throughout the duration of the test.


Description of key information

No acute EC50 is determined in freshwater and marine water invertebrates up to solubility limit.

Key value for chemical safety assessment

Additional information

Four studies are available in fresh water and two in sea water.

Three Daphnia magna studies (Scholz 1990 & 1994, Knacker 1990) are considered as not reliable as they make use of a surfactant to expose the animals at concentrations well above solubility limit. In addition the surfactant concentration varies with the test substance concentration which does not allow to separate effects of the two. In the fourth Daphnia test (Thiébaud, 1996), no immobilization was observed after 48h exposure at the highest soluble (measured) concentration, 29 µg/L.

In the first Acartia test (Rogaland 1996) a LC50 -48h has been determined as being 87 µg/L (nominal), using acetone as solvent.

In the second Acartia test (Inveresk, 1997) LC50 was not attained at the highest soluble concentration of 20.6 µg/L.

It can be concluded that in normal conditions (not trespassing water solubility limit) there is no significant acute toxicity towards fresh- or seawater invertebrates.