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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:
key study
Study period:
from 01 aug 2006 to 10 aug 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to satndardised guidelines and GLP.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: just before the start of the test: duplicate samples of each test medium (without daphnids) and duplicate samples from the control (without daphnid) were taken for analysis. After 48 hours (from the stability samples): duplicate samples from each test medium and duplicate samples from the control were also taken for analysis. However only the concentrations of the test item Paradimethoxybenzene were analyzed in one of the duplicate test medium samples of the dilutions 1:4, 1:2 and of the undiluted filtrate from both sampling times (0 and 48 hours) were anlysed, the samples taken from the three lowest test concentrations (dilutions 1:8, 1:16 and 1:32) were not analyzed, since these concentrations were below the 48 hour NOEC, determined in this test. From the control samples, one of the duplicate samples was analyzed from each of the sampling times.

- Sampling method: no data

- Sample storage conditions before analysis: All samples were deep-frozen (at about -20 °C) immediately after sampling and protected from light until analysis was performed. The stability of the frozen samples was tested during non-GLOP pre-exepriments
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
The test media were prepared as follows: The supersaturated dispersion with a loading rate of 100 mg/L was prepared by weighing 61.3 mg of the
test item into 600 mL of test water. No auxiliary solvent or emulsifier was used. The test item was mixed into the test water as homogeneously as
possible using ultrasonic treatment for 15 minutes and intense stirring for 3 hours at room temperature in the dark to dissolve a maximum
concentration of the test item in the dispersion. Then, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm). The filtrate was used as the highest concentrated test medium and as a stock solution for the preparation of the test media with lower test item concentrations (dilutions 1:2, 1:4, 1:8, 1:16 and 1:32).The test media were prepared just before introduction of daphnids.

- Eluate: no
- Differential loading: no
- Controls: Additionally, a control (test water without test item) was tested in parallel
- Chemical name of vehicle: none
- Evidence of undissolved material: according to the results of a pre-experiment (without GLP), the test item was not completely soluble at the
concentration of 100 mg/L in the test water and no homogeneous dispersion could be prepared.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: daphnia species
- Strain: Daphnia magna Straus clone 5
- Source: originally supplied supplied by the University of Sheffield/UK in 1992.
- Age at study initiation: 6-24 hours old
- Method of breeding: the clone is bred in the laboratories of RCC in reconstituted water of the quality identical to the water quality used in the tests
(in respect of pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests
- Feeding during test: no

ACCLIMATION: not applicable

QUARANTINE (wild caught): not applicable
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
no data
Hardness:
250 mg/L CaCO3
Test temperature:
20°C
pH:
7.8 - 7.9
Dissolved oxygen:
8.6 - 8.9 mg/l
Salinity:
no data
Nominal and measured concentrations:
Nominal: The supersaturated dispersion with a loading rate of 100 mg/L , filtrated and used as the highest concentrated test medium and as a stock solution for the preparation of the test media with lower test item concentrations (dilutions 1:2, 1:4, 1:8, 1:16 and 1:32).
Measured: The average concentrations found in the test medium samples at test start were found to be 19.4 mg/L (Filtrate 1:4), 40.0 mg/L (Filtrate 1:2) and 78.3 mg/L (Filtrate undiluted) and at test end 14.8 mg/L (Filtrate 1:4), 29.3 mg/L (Filtrate 1:2) and 58.2 mg/L (Filtrate undiluted).
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: 100 mL glass beakers filled with 50 mL of test medium
- Aeration: prior to the start of the study until oxygen saturation was reached. During the test period the test water was not aerated.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: one daphnia per 10 ml

WATER PARAMETERS
- Source/preparation of dilution water:
Reconstituted test water: analytical grade salts were dissolved in purified water to obtain the following nominal concentrations:

CaCl2,2H2O: 2.0 mmol/L (=294 mg/L)
MgSO4,7H2O: 0.5 mmol/L (=123 mg/L)
NaHCO3: 0.75 mmol/L (=65 mg/L)
KCl: 0.075 mmol/L (=5.8 mg/L)
Water Hardness: 2.5 mmol/L (=250 mg/L as CaCO3)
Alkalinity: 0.8 mmol/L
Ratio of Ca : Mg = 4 : 1 (based on molarity)
Na : K = 10 : 1 (based on molarity)
- Culture medium different from test medium: no
- Intervals of water quality measurement: At the start and at the end of the test, the pH values, the dissolved oxygen concentrations and the water temperature were determined in each test concentration and the control. The appearance of the test media was recorded at the start of the test and after 24 and 48 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A 16-hour light to 8-hour dark photoperiod with a 30 minute transition period
- Light intensity: 470-640 Lux


EFFECT PARAMETERS MEASURED:
The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure. Those daphnids that were not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilized.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: undiluted filtrate of a saturated solution and dilutions 1/10, 1/100 and 1/1000.
- Results used to determine the conditions for the definitive study: No effects were observed at the dilutions 1/1000 - 1/10. At the undiluted filtrate, 70% and 100% of the daphnids were immobilized after 24 and 48 hours respectively.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
52 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 49-55
Details on results:
- Behavioural abnormalities: no data
- Observations on body length and weight: not applicable
- Other biological observations: no data
- Mortality and Other adverse effects in control: none of the control daphnids showed immobilization or other signs of disease or stress.
- Abnormal responses: no data
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: not applicable
Results with reference substance (positive control):
The latest result of the positive control test in June 2006 (48 hour EC50: 0.67 mg/L, RCC Study No. A72753) showed that the sensitivity of the test organisms was within the historical range of the RCC laboratory (48-hour EC50 from 1996 to 2006: 0.53-1.1 mg/L).
- Results with reference substance valid? Yes
Reported statistics and error estimates:
The 24-hour EC50 could not be calculated by Probit Analysis or Moving Average Interpolation due to the low toxicity of the test item at the highest test concentration after 24 hours. Instead, the 24-hour EC50 was calculated by linear interpolation of the immobility between the two consecutive test concentrations of 34 and 68 mg/L on a semi-logarithmic scale (the 95% confidence limits could not be determined).

The 48-hour EC50 and the 95% confidence limits were calculated by Probit Analysis (Ref. 1, 2). The biological results obtained for the three lowest test concentrations (dilutions 1:8, 1:16 and 1:32) were not taken into account at the Probit Analysis, because these test concentrations were below the 48-hour NOEC, and thus not analyzed. The calculations were based on the mean measured test item concentrations (see the discussion of the analytical results).

The NOEC and EC0 were determined directly from the raw data. The 24- and 48-hour EC100 of the test item could not be determined directly from the raw data because of the low toxicity of the test item.

See table

Dilution / Treatment

Mean measured concentration

No. of
daphnids
tested

Immobilized
daphnids after
24 hours

Immobilized
daphnids after
48 hours

(mg/L)

No.

%

No.

%

Control

---

20

0

0

0

0

1:32

n.a.

20

0

0

0

0

1:16

n.a.

20

0

0

0

0

1:8

n.a.

20

0

0

0

0

1:4

17

20

0

0

0

0

1:2

34

20

1

5 *

2

10

Undiluted filtrate

68

20

11

55

16

80

n.a.:     not analyzed

*:         An immobilization rate of 10% is tolerated by the test guidelines



Validity criteria fulfilled:
yes
Conclusions:
Harmful for aquatic organisms
Executive summary:

The acute toxicity of the test item Paradimethoxybenzene to Daphnia magna was determined in a 48 hour static test according to the EU Commission Directive 92/69/EEC, Part C.2 (1992) and the OECD Guideline for Testing of Chemicals, No. 202 (2004).

Due to the low water solubility of the test item, a supersaturated dispersion of the test item with a loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 3 hours. Then, the dispersion was filtered. The undiluted filtrate of the dispersion and dilutions 1:2, 1:4, 1:8,
1:16 and 1:32 were used as test media. Additionally, a control was tested in parallel.

At the start of the test, the analytically measured test item concentrations in the analyzed test media (dilutions 1:4, 1:2 and the undiluted filtrate) amounted to 19, 40 and 78 mg/L, respectively. In these test media, the measured concentrations of Paradimethoxybenzene decreased to values of 15, 29 and 58 mg/L over 48 hours. The reported biological results are based on the mean measured (calculated as the geometric mean over all measurements in the test medium) test item concentrations. The tabulated values represent rounded results obtained by calculation using the exact raw data.

After 48 hours of exposure, no immobility of daphnids was observed in the control and at test item concentrations up to and including 17 mg/L. At the highest test item concentration of 68 mg/L, 80% of the daphnids were immobile.

The 48-hour EC50 was calculated to be 52 mg/L with 95% confidence limits of 49 and 55 mg/L. The 48-hour EC0 and also the 48-hour NOEC (highest concentration tested without toxic effects after 48 hours) of Paradimethoxybenzene were 17 mg/L, since no immobilization was observed in the test organisms up to and including this test
concentration.

Based on the results of this study, Paradimethoxybenzene would  be classified as harmful to aquatic organisms, according to the EU classification criteria.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: The French registered guideline T90301 (January 1983) is an official testing guideline and it can be supposed that the source of p-Dimethoxybenzene is Rhone Poulenc (purity not determined).
Qualifier:
equivalent or similar to
Guideline:
other: AFNOR T 90301 Standard, French
Deviations:
not specified
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
no data
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 100 mg of test substance was introduced in 1 L of synthetic river water (composition according to the guideline) and this solution was
mixed during 30 minutes by ultrasonification. The mixed solution was then filtered on PTFE membrane of 0.22 µ.
- Eluate: no data.
- Differential loading: no data
- Controls: no data.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not used
- Concentration of vehicle in test medium (stock solution and final test solution): not used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: daphnia species
- Strain: Daphnia magna
- Source: no data
- Age at study initiation (mean and range, SD): no data
- Method of breeding: no data
- Feeding during test: no data

ACCLIMATION: no data

QUARANTINE (wild caught): no data
Test type:
other: probably static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
24 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
20°C
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
no data
Details on test conditions:
TEST SYSTEM: no data

TEST MEDIUM / WATER PARAMETERS: no data

OTHER TEST CONDITIONS: no data


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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: water saturated at 100 mg/l of PDMB, sonicated and then filtrated. Dilutions from this saturated solution. Only 100% and 35% solutions are reported

Reference substance (positive control):
not specified
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
35 - 100 other: %
Nominal / measured:
nominal
Conc. based on:
other: % of the saturated solution
Basis for effect:
mobility
Remarks on result:
other: not determined
Details on results:
EC50-24 hours was between 35 and 100% of saturated solution.
25% of inhibition was obtained with 35% of saturated solution.
100% of inhibition was obtained with 100% of saturated solution.
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
not determined
Validity criteria fulfilled:
not specified

Description of key information

Daphnia magna were exposed in static conditions to paradimethoxybenzene according to OECD 202 and GLP. The 48-hour EC50 was calculated to be 52 mg/L ( based on mean measured test item concentrations calculated as the geometric mean over all measurements per test concentration during the test period).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
52 mg/L

Additional information

Only two studies were available. One of them (RCC, 2006) quoted as reliability 1 according to Klimisch, was selected as key study; the other one of reliability 4 (Palla, 1983) was selected as supporting study.