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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:
1993-04-28 till 1993-07-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
(1984)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
(1984)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Concentrations of 0.75, 1.5, 3, 6 and 12 mg/L were measured at 0 and 48 hours; and concentration of 0 mg/L at 48 hours only.
- Sample storage conditions before analysis: Routinely, the sample were analysed immediately. 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene was stored in the dark at ambient temperature until required.
Vehicle:
no
Details on test solutions:
Preparation of dilutions of the test material
A stock dilution, nominally containing 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene at 12 mg/L, was prepared by adding the required weight of material, mixed with acetone, to dilution water: to aid dissolution it was treated by ultrasound for ten minutes. The stock was either used directly or further diluted to provide the test media.
Test organisms (species):
Daphnia magna
Details on test organisms:
Test organism
The strain of Daphnia magna used in this study was obtained from the University of Sheffield where electrophoretic assay had confirmed genetic homogeneity.
Daphnia have been maintained in parthenogenetic culture at the Aquatic Studies Laboratories of Pharmaco-LSR since 3 August 1989.

Daphnia culture method
The culture vessels were two-litre Pyrex glass beakers with loose-fitting clock glass covers.
The cultures were kept in a temperature-controlled laboratory nominally maintained at 20 + 2·c. The day length in the area was controlled giving a photoperiod of 16 hours light, supplied by overhead fluorescent tubes, and eight hours darkness. Dawn and dusk were simulated by a period of subdued lighting at·the beginning and end of the light phase.
The water (1.5 litres) in each culture vessel was replaced at intervals of two weeks with fresh water of the correct temperature. At no stage of routine culture were Daphnia transferred between solutions differing in temperature by more than 2 °C.
A maximum of twenty adult Daphnia were maintained in each culture vessel and juveniles which were produced were removed at least twice each week.

Culture feeding regime
Daphnia cultures were fed at least five times each week with suspensions of the unicellular green alga Chlorella vulgaris and yeast.
Chlorella vulgaris, strain CCAP 211/12 obtained from the Culture Centre of Algae and Protozoa {CCAP, The Freshwater Biological Association, Cumbria, England), was cultured in a synthetic mineral salts medium in illuminated ten-litre glass fermenter vessels.
Concentrated algal cell suspensions were prepared by removing and centrifuging aliquots of cell suspension from the fermenter and re-suspending the resultant pellets of algae in small volumes of culture medium. Appropriate volumes of these concentrated cell suspensions were added to each Daphnia culture to give a resulting density in each culture vessel of 2 to 8 x 10 cells per mL.
A suspension (100 mg/L) of dried baking yeast was prepared each week in culture medium and an aliquot (0.6 mL) was added to each Daphnia culture to give a nominal concentration of 0.04 mg/L in the culture vessel.

Procedure for obtaining juvenile Daphnia
Before the start of each test, gravid (i.e. egg-bearing) Daphnia were isolated from laboratory cultures and held in water taken from the culture vessel. Isolated Daphnia were fed each day at a rate equivalent to the feeding level in culture. Juveniles produced by the isolated Daphnia were removed each day. Juvenile Daphnia that were to be used in a test were removed on the day of the test so as to be between 6 and 24 hours old when used. Between the time of isolation and use in the definitive test, the juveniles were held in water at 19.3 to 19.7 °C.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
208 - 211 mg CaCO3/L = 11.7 - 11.8 °dH
Test temperature:
17.5 - 19.5 °C
pH:
7.6 - 8.4
Dissolved oxygen:
96-100 % ASV
Nominal and measured concentrations:
nominal: 0.75, 1.5, 3, 6 and 12 mg/L
measured: 0.743, 1.5, 3.1, 6.24 and 12.6 mg/L
Details on test conditions:
Test procedure
The tests were carried out under static exposure conditions.
A preliminary, rangefinding test was conducted in which Daphnia were exposed for 48 hours to 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene at nominal concentrations of 0.01, 0.1, 1.0, 10 and 100 mg/L.
Based on the results of this test, the definitive test employed nominal exposure concentrations of 0.75, 1.5, 3, 6 and 12 mg/L.
Control groups of Oaphnia were placed in dilution water alone or dilution water containing acetone at the same level as in the test dilutions (0.1 ml/1). Four vessels, each containing five animals, were employed for each test and control group.
Daphnia were removed from the holding vessel using a pipette and allocated, in groups of five, to the test vessels according to random numbers. The test vessels were arranged in the test area in the same order as the Daphnia were added to the test vessels.
The time between adding the test material to water and adding the last batch of Oaphnia to the test vessels was 120 minutes.
The test dilutions were not aerated during the test. Their pH was not adjusted nor controlled.
Observations of the Daphnia were made 24 and 48 hours after the start. The appearance of the test dilutions was noted at the start and end of the test.
The temperature, pH and concentration of dissolved oxygen (DO) of the dilution water and preparations of test material at each exposure concentration were measured at the start and end.
Temperature, pH and DO measurements were made using the test dilutions remaining in the preparation flasks after the test vessels were filled at the start of the test, and in additional vessels which contained no Daphnia at the end.
The total hardness and alkalinity of the dilution water were measured at the start and end of the test.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
2.69 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (2.14 - 3.33 mg/LL)
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.06 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (1.67 - 2.53 mg/L)
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.743 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The 48-hour median effect concentration (EC50) for immobility, based on measured concentrations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene in unfiltered media at the start, was found to be 2.06 mg/L (95 % confidence limits of 1.67 and 2.53 mg/L).

The results of chemical analysis show that intended exposure levels were achieved but they decreased (by > 90 %) during the test indicating that 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene was not stable in the test dilution water at the concentrations employed. Since the test material is not volatile and such extensive loss is unlikely to be the result of adsorption, the observed decrease in exposure levels is thought to be due to the hydrolytic instability of the test material. Daphnia were therefore exposed to both the parent material and its hydrolysis products during the course of the test.

Results indicate that intended exposure concentrations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene were achieved {between 99 and 105 % of the nominal values in unfiltered media). The levels in filtered media were slightly lower (between 84 and 88 % of their nominal values): since most test concentrations were within the aqueous solubility range of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene, this suggested that some material may have been lost by adsorption during the filtration process.

After 48 hours, measured levels in all samples had decreased (to between <1 and 3 % of their nominal values): these results suggest that at the concentrations used in the test, 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene is unstable in the test dilution water.

In the calculation of the test results, exposure concentrations have been based on the measured levels in unfiltered samples at the start of the test
(0.743, 1.5, 3.1, 6.24 and 12.6 mg/L).

Analytical determinations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene

 Nominal

Measured 1,3 -bis(3 -methyl-2,5 -dioxo-1H-pyrrolinylmethyl)benzene concentrations (mg/L)

 concentration     0 hours     48 hours
 (mg/L)  uf  f  uf  f
 0  nd  nd  nd  nd
 0.75  0.743 (99)  0.641 (85)  0.00841 (1)  nd
 1.5  0.286*  0.264*  0.0167 (1)  0.00556 (<1)
 3  3.10 (103)  2.60 (87)  0.0230 (<1)  0.0277 (<1)
 6  6.24 (104)  5.03 (84)  0.106 (3)  0.0497 (<1)
 12  12.6 (105)  10.5 (88)  0.303 (3)  0.199 (2)

nd: None detected (< 0.005 mg/L)

( ): Measured concentration expressed as a percentage of the nominal concentration.

*: These values are anomalously low and have been excluded from the test calculations. Although the source of the error has not been identified, the measured levels at 48 hours are consistent with those found at the adjacent concentrations. Therefore it is considered that the test medium was correctly formulated.

uf: unfiltered media

f: filtered media

Observations of the numbers of mobile, immobile, submerged and floating Daphnia

 Measured test

          Number of Daphnia

concentrations (mg/L

    Mobile

    Immobile

 and observation times

 submerged

 floating

 submerged

 floating

 24 hours        
 control (water)  20  0  0  0
 control (acetone)  20  0  0  0
 0.743  20  0  0  0
 1.5*  19  0  1  0
 3.1  0  0  20  0
 6.24  8  0  10  2
 12.6  0  0  20  0
 48 hours        
 control (water)  19  1  0  0
 control (acetone)  20  0  0  0
 0.743  19  1  0  0
 1.5*  19  0  1  0
 3.1  0  0  20  0
 6.24  1  0  19  0
 12.6  0  0  20  0

* At 1.5 mg/L, the nominal value has been used because the measured levels were anomalously low at the start of the test.

Validity criteria fulfilled:
yes
Remarks:
(-The immobilisation and other abnormalities in the controls did not exceed 10 % by the end of the Test. -The dissolved oxygen concentration remained above 3 mg/L throughout the exposure period.)
Conclusions:
Daphnia magna was exposed under static conditions for 48 hours. The concentrations (0.743, 1.5, 3.1, 6.24 and 12.6 mg/L) of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene were measured in unfiltered samples. An EC50 of 2.06 mg/L was determined and a NOEC of 0.743 mg/L was calculated.
Executive summary:

In order to test acute toxicity to inverbrates of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene, daphnia magna was exposed to the test solution of five nominal concentrations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene (0.75, 1.5, 3, 6 and 12 mg/L) and blank control solution for a period of 48 hours under static conditions. Mobility and visible abnormalities were recorded at 24 and 48 hours.

Exposure levels were monitored by an HPLC method of chemical analysis; the limit of this assay was estimated to be 0.005 mg/L. The concentrations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene in solution (determined after filtration: 0.2 µm pore size) and suspension were measured in duplicate mid-vessel samples taken at each exposure level at the start and end of the test.

Results indicate that intended exposure concentrations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene were achieved (between 99 and 105 % of the nominal values in unfiltered media). The levels in filtered media were slightly lower (between 84 and 88 % of their nominal values): since most test concentrations were within the aqueous solubility range of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene, this suggested that some material may have been lost by adsorption during the filtration process. After 48 hours, measured levels in all samples had decreased (to between <1 and 3 % of their nominal values): these results suggest that at the concentrations used in the test, 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene is unstable in the test dilution water.

In the calculation of the test results, exposure concentrations have been based on the measured levels in unfiltered samples at the start of the test (0.743, 1.5, 3.1, 6.24 and 12.6 mg/L).

The 48-hour median effect concentration (EC50) for immobility, based on measured concentrations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene in unfiltered media at the start, was found to be 2.06 mg/L.

The highest measured concentration at which no immobilisation occurred and the lowest at which there was 100 % immobility after 48 hours was 0.743 and 3.1 mg/L respectively. The no-observed-effect concentration (NOEC) was thus 0.743 mg/L.

The results of chemical analysis show that intended exposure levels were achieved but they decreased (by > 90 %) during the test indicating that 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene was not stable in the test dilution water at the concentrations employed. Since the test material is not volatile and such extensive loss is unlikely to be the result of adsorption, the observed decrease in exposure levels is thought to be due to the hydrolytic instability of the test material. Daphnia were therefore exposed to both the parent material and its hydrolysis products during the course of the test.

Description of key information

Daphnia magna was exposed under static conditions for 48 hours. The concentrations (0.743, 1.5, 3.1, 6.24 and 12.6 mg/L) of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene were measured in unfiltered samples.  An EC50 of 2.06 mg/L was determined and a NOEC of 0.743 mg/L was calculated.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
2.06 mg/L

Additional information

In order to test acute toxicity to inverbrates of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene, daphnia magna was exposed to the test solution of five nominal concentrations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene (0.75, 1.5, 3, 6 and 12 mg/L) and blank control solution for a period of 48 hours under static conditions. Mobility and visible abnormalities were recorded at 24 and 48 hours.

Exposure levels were monitored by an HPLC method of chemical analysis; the limit of this assay was estimated to be 0.005 mg/L. The concentrations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene in solution (determined after filtration: 0.2 µm pore size) and suspension were measured in duplicate mid-vessel samples taken at each exposure level at the start and end of the test.

Results indicate that intended exposure concentrations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene were achieved (between 99 and 105 % of the nominal values in unfiltered media). The levels in filtered media were slightly lower (between 84 and 88 % of their nominal values): since most test concentrations were within the aqueous solubility range of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene, this suggested that some material may have been lost by adsorption during the filtration process. After 48 hours, measured levels in all samples had decreased (to between <1 and 3 % of their nominal values): these results suggest that at the concentrations used in the test, 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene is unstable in the test dilution water.

In the calculation of the test results, exposure concentrations have been based on the measured levels in unfiltered samples at the start of the test (0.743, 1.5, 3.1, 6.24 and 12.6 mg/L).

The 48-hour median effect concentration (EC50) for immobility, based on measured concentrations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene in unfiltered media at the start, was found to be 2.06 mg/L.

The highest measured concentration at which no immobilisation occurred and the lowest at which there was 100 % immobility after 48 hours was 0.743 and 3.1 mg/L respectively. The no-observed-effect concentration (NOEC) was thus 0.743 mg/L.

The results of chemical analysis show that intended exposure levels were achieved but they decreased (by > 90 %) during the test indicating that 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene was not stable in the test dilution water at the concentrations employed. Since the test material is not volatile and such extensive loss is unlikely to be the result of adsorption, the observed decrease in exposure levels is thought to be due to the hydrolytic instability of the test material. Daphnia were therefore exposed to both the parent material and its hydrolysis products during the course of the test.