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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

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

Remarks:

Guideline study performed under GLP including analytical monitoring. There are some minor deficiencies concerning the analytical results between filtered and unfiltered samples. Fish have been placed in filtered samples but calculation of effect values was done on unfiltered results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

(1984)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

(1984)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Concentrations of 63, 125, 250, 500 and 1000 µg/L were measured at 0, 24, 72 and 96 hours.
- Sample storage conditions before analysis: The sample of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene was contained in a plastic jar; this was stored in the dark at ambient temperature until required.

Vehicle:

no

Details on test solutions:

Groups of ten fish were exposed to 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene at nominal concentrations of 63, 125, 250, 500 and 1000 µg/L, selected following two preliminary, rangefinding tests.
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 and suspension were measured in filtered (0.2 µm pore size) and unfiltered mid-vessel samples which were taken from two sets of freshly prepared and old (24 hours) media.
Results indicate that intended exposure concentrations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene were reasonably well achieved (between 74 and 91 % of the nominal values in unfiltered media). Levels in filtered samples were lower (between 62 and 76 % of the nominal values), and since it is believed that all test concentrations were within the aqueous solubility range of the substance. This suggests that some test material may have been lost by adsorption during the filtration process.
In the calculation of the test results, exposure concentrations have been based on the overall mean measured levels in unfiltered samples (34.8, 65.9, 132, 267 and 599 µg/L).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Test organism
The batch of fish used for the definitive test was supplied by Fish Network Limited, Devon. The fish were reared at Exmoor Trout Farm, Devon from English eggs which hatched late December 1992.
The fish were transported to Pharmaco-LSR in aerated water taken from the farm, on 16 February 1993. The hardness of the water
used to transport the fish, determined at the Laboratory on the day of delivery, was 246 mg/L as CaC03 (= 13.8 °dH).
At the laboratory, these fish were held in an aerated supply of dilution water under flow-through conditions until removed for use in tests.
Monitoring water quality during the 14-day holding period immediately preceding the definitive test showed the following:
- temperatures ranged from 12.4 to 14.3 °C
- pH values ranged from 7.8 to 8.3
- dissolved oxygen concentrations (DO) ranged from 92 to 102 % air saturation value (ASV)
- water hardness ranged from 234 to 240 mg/L as CaC03 (= 13.1 to 13.4 °dH).
Each day during the holding period, the fish were fed with proprietary trout pellets, an amount equivalent to approximately 1 to 4 % of the total wet-weight of the fish in the holding tank.
Mortality during the 14-day period before the definitive test was less than 0.5 %.
The fish were last fed approximately 26 hours before the start of the definitive test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

228 to 252 mg CaCO3/L = 12.8 to 14.1 °dH

Test temperature:

14.0 to 14.9 °C

pH:

7.6 to 8.2

Dissolved oxygen:

97-100 % of saturation

Nominal and measured concentrations:

nominal concentrations: 62.5, 125, 250, 500, 1000 µg/L
measured concentrations: 34.8, 65.9, 132, 267 and 599 µg/L

Details on test conditions:

Apparatus
The test vessels were all-glass aquaria, with a total capacity of 15 litres. The depth of the test solutions in these vessels was approximately 13 cm.
Aeration of the contents of each vessel was achieved using a Pasteur pipette connected to an oil-free supply of compressed air.

Preparation of dilutions of the test material
Two freshly-prepared concentrated aqueous dispersions, each nominally containing 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene at
5 mg/L, were prepared in test dilution water: to aid dissolution they were treated by ultrasound for ten minutes before use. One of these was diluted to provide the test medium at the highest concentration (1000 µg/L); aliquots of the other were used to prepare the remaining test media (63 to 500
µg/L).

Test procedure
Two preliminary, rangefinding tests were conducted under static conditions in which fish were exposed to 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene at nominal concentrations of 1, 10, 100 and 1000 mg/L for 24 hours and to 10, 100 and 1000 µg/L for 96 hours. In both tests, ultrasound treatment was used during the preparation of the test media; in the first test, acetone was also used to aid dissolution of the test material but, in the second test because the exposure levels were within the aqueous solubility of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene (64 mg/L, information supplied by the Sponsor), it was not considered necessary.
Based on the results of these tests, the definitive test was conducted under semistatic conditions with replacement of the test and control media every 24 hours. A flow-through regime was considered but not thought to be appropriate because of the instability of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene in water and the technical difficulties in achieving sufficient accuracy when dosing the test material from a· concentrated solvent solution, without exceeding the recommended level of solvent (0.1 mL/L). Nominal exposure concentrations of 62.5, 125, 250, 500 and 1000
µg/L were employed. A control group of fish was exposed to dilution water alone.
The fish were allocated, in groups of five, to the vessels containing the prepared test or control media until each contained ten animals.
At 24, 48 and 72 hours, the surviving fish in each vessel were transferred to another containing fresh dilution water or test medium.
The test and control media were gently aerated during the test. Their pH was not adjusted nor controlled. The appearance of the test dilutions was noted during the test.
Observations of the fish were made after 24, 48, 72 and 96 hours; additional observations were made during the first four hours. The fish were not fed during the test.
The temperature, pH and concentration of dissolved oxygen of the contents of each vessel were measured at the start of the test and thereafter each day either immediately before or following the observations of fish behaviour. The total hardness of the dilution water control and selected test dilutions was determined at the start and end.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.188 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

0.188 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.174 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.066 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Sublethal observations / clinical signs:

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

nominal conc.		Measured concentration, µg/L
µg/L		0 hours		24 hours		72 hours		96 hours		overall mean
		new		old		new		old
62.5	uf	50.0	(80)	19.4	(31)	53.1	(85)	16.8	(27)	34.8	(56)
	f	39.8	(64)	16.9	(27)	41.2	(66)	13.9	(22)	28.0	(45)
125	uf	92.3	(74)	31.5	(25)	111	(89)	28.9	(23)	65.9	(53)
	f	91.8	(73)	26.3	(21)	83.5	(67)	22.8	(18)	56.1	(45)
250	uf	192	(77)	71.5	(29)	193	(77)	70.5	(28)	132	(53)
	f	154	(62)	60.5	(24)	159	(64)	53.3	(21)	107	(43)
500	uf	395	(79)	139	(28)	*		*		267	(53)
	f	351	(70)	144	(29)	*		*		248	(50)
1000	uf	913	(91)	285	(29)	*		*		599	(60)
	f	761	(76)	269	(27)	*		*		515	(52)

uf = unfiltered sample.

f = filtered sample.

* = no samples taken because all of the fish at these levels had died.

( ) = measured concentration expressed as a percentage of the nominal value.

Cumulative mortality

Observation times	Measured concentration, µg/L
(hours)	0	34.8	65.9	132	267	599
2	0	0	0	0	0	0
4	0	0	0	0	0	0
24	0	0	0	0	0	10
48	0	0	0	0	10	10
72	0	0	0	0	10	10
96	0	0	0	1	10	10

Validity criteria fulfilled:

yes

Remarks:

(- The mortality in the controls did not exceed 10 % by the end of the test. - The dissolved oxygen concentration remained above 60 % of the air-saturation value throughout the exposure period. -The pH did not vary by more than 1 unit.)

Conclusions:

The acute toxicity to fish (rainbow trout) was tested in a semi-static test. After 96 hours of exposure in unfiltered sample a LC50 of 174 µg/L was measured and a NOEC of 65.9 µg/L was calculated.

Executive summary:

A study was performed to assess the acute toxicity of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene to Rainbow trout under semi-static conditions.

The study was conducted in accordance with Official Journal of the European Communities. Method C l "Acute toxicity for fish" (1984.) which is in most parts equivalent to the OECD Guidelines for Testing of Chemicals No. 203 "Fish, Acute Toxicity Test" (1984).

Concentrations of 63, 125, 250, 500 and 1000 µg/L (nominal) were measured at 0, 24, 72 and 96 hours.

After 96 hours of exposure in unfiltered sample a LC50 of 174 µg/L was measured and a NOEC of 65.9 µg/L was calculated.

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 and suspension were measured in filtered

(0.2 µm pore size) and unfiltered mid-vessel samples which were taken from two sets of freshly prepared and old (24 hours) media.

Results indicate that intended exposure concentrations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene were reasonably well achieved (between 74 and 91 % of the nominal values in unfiltered media). Levels in filtered samples were lower (between 62 and 76 % of the nominal values), and since it is believed that all test concentrations were within the aqueous solubility range of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene this suggests that some test material may have been lost by adsorption during the filtration process.

In 24-hour-old media, measured levels in all samples had decreased (to between 18 and 31 % of their nominal values), suggesting 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 overall mean measured levels in unfiltered samples (34.8, 65.9, 132, 267 and 599 µg/L).

The results of chemical analysis show that intended exposure levels were reasonably well achieved but decreased (by 69 to 82 %) between daily renewals of test media 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. This instability was probably the result of hydrolysis because the test material is not volatile and the loss of the parent test material was too extensive and independent of its nominal concentration for adsorption to have been the cause. Therefore, it is considered that the fish were exposed to both the parent material and its hydrolysis products throughout the test.

Description of key information

The acute toxicity to fish (rainbow trout) was tested in a semi-static test.

After 96 hours of exposure in unfiltered sample a LC50 of 174 µg/L was measured and a NOEC of 65.9 µg/L was calculated.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

174 µg/L

Additional information

A study was performed to assess the acute toxicity of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene to Rainbow trout under semi-static conditions.

The study was conducted in accordance with Official Journal of the European Communities. Method C l "Acute toxicity for fish" (1984.) which is in most parts equivalent to the OECD Guidelines for Testing of Chemicals No. 203 "Fish, Acute Toxicity Test" (1984).

Concentrations of 63, 125, 250, 500 and 1000 µg/L (nominal) were measured at 0, 24, 72 and 96 hours.

After 96 hours of exposure in unfiltered sample a LC50 of 174 µg/L was measured and a NOEC of 65.9 µg/L was calculated.

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 and suspension were measured in filtered

(0.2 µm pore size) and unfiltered mid-vessel samples which were taken from two sets of freshly prepared and old (24 hours) media.

Results indicate that intended exposure concentrations of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene were reasonably well achieved (between 74 and 91 % of the nominal values in unfiltered media). Levels in filtered samples were lower (between 62 and 76 % of the nominal values), and since it is believed that all test concentrations were within the aqueous solubility range of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene this suggests that some test material may have been lost by adsorption during the filtration process.

In 24-hour-old media, measured levels in all samples had decreased (to between 18 and 31 % of their nominal values), suggesting 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 overall mean measured levels in unfiltered samples (34.8, 65.9, 132, 267 and 599 µg/L).

The results of chemical analysis show that intended exposure levels were reasonably well achieved but decreased (by 69 to 82 %) between daily renewals of test media 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. This instability was probably the result of hydrolysis because the test material is not volatile and the loss of the parent test material was too extensive and independent of its nominal concentration for adsorption to have been the cause. Therefore, it is considered that the fish were exposed to both the parent material and its hydrolysis products throughout the test.