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

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September - October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study according to protocol. Although males were present in the test the results can still be used for classification and risk assessment as the females present performed well and if this fact would have an impact on the results this is negative (the animals will have a worse condition and will therefore be mroe sensitive) and the endpoint are lower. Therefore this study can be seen as a worst case.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

The temperature of culture water and test water did not always fall within the described limits.

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Name of test material: Perkalink 900
Lot No.: 812191
purity: 92.3%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

water samples were removed from two replicates of each treatment level, control and solvent control and analyzed for Perkalink 900 concentration on test days 0, 7, 11, 14, 18 and 21. All samples were removed from the approximate mid-point of the appropriate vessels.
The test solutions were sampled and analyzed on day 7. However, the results of this analysis were unsatisfactory and furthermore not consistent with the results obtained at the other sampling points. Therefore these results were deemed to be unreliable and were not used for further calculations.
In addition, three quality control (QC) samples and one blank control were prepared at each sampling interval and remained with the set of exposure solution samples throughout the analytical process.

Test solutions

Vehicle:

yes

Details on test solutions:

For the 0.064, 0.16, 0.4 and 1.0 mg test item/L test concentrations, stock solutions in acetone used for the diluter system were prepared seven days prior to test initiation and were stored under refrigerated conditions until use.
For the lowest test concentration, a stock solution with a concentration of 0.26 mg test item/mL was prepared by diluting 2.6 mL of the 10 mg test item/mL stock solution to 100 mL using acetone.
For the solvent control, a stock solution containing only acetone was prepared. For the
dilution water control only dilution water without test item and solvent was used.
During testing, all stock solutions were stored at room temperature.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The Daphnia magna used in this toxicity test were obtained from laboratory cultures maintained at Springborn Smithers Laboratories (Europe) (new Smithers Viscient AG), originally obtained from a culture at Springborn Smithers Laboratories Wareham (MA) (new Smithers Viscient LLC), USA.
Prior to testing, the daphnids were held in glass beakers under a photoperiod of 16 hours light and 8 hours darkness with a 30 minute transition period. The culture water was Elendt M4 medium, prepared of well water from the municipality of Horn, deionized with a Culligan Reverse Osmosis system.
The daphnids (<24 hours old) used during the definitive exposure were maintained under the following conditions for two days prior to testing:
Total Hardness (mg/L CaCO3) 148
Total Alkalinity (mg/L CaCO3) 24
Specific Conductivity (μS/cm) 350
pH 7.70
Temperature (°C) 16 – 21
Daphnids were fed 2.0 mL of a solution containing approximately 4 x 107 cells/mL of the unicellular green alga, Ankistrodesmus falcatus (ANK) and approximately four drops of a combination of yeast, cereal leaves and flaked fish food (YCT) daily prior to the definitive exposure.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

146 to 156 mg/L CaCO3

Test temperature:

The single point temperature was between 19.5 to 22.9ºC, whereas the continuously measured temperature ranged from 18.3 to 22.1°C throughout the exposure.

pH:

7.06 to 7.93

Dissolved oxygen:

3.70 to 10.25 mg/L

Nominal and measured concentrations:

Nominal concentrations: 0.026, 0.064, 0.16, 0.4 and 1.0 mg test item/L
Measured: time weighted mean concentrations were 0.017, 0.057, 0.151, 0.334 and 0.800 mg test item/L.

Details on test conditions:

The batches of Elendt M4 medium used for the definitive test had a total hardness range from 146 to 152 mg/L as CaCO3, an alkalinity range from 27 to 31 mg/L as CaCO3, a pH range from 6.74 to 7.47 and a specific conductivity range from 315 to 390 μS/cm.

The toxicity test was conducted using an exposure system consisting of a modified proportional diluter (Mount and Brungs, 1967), a temperature-controlled water bath and a set of 28 exposure vessels. The exposure system was designed to provide five concentrations of the test item, a dilution water control and a solvent control. The aquaria were randomly positioned in a water bath containing circulating water designed to maintain the test solution temperatures at 20 ± 1°C. The test was illuminated to a light intensity of 200 to 500 lux using fluorescent bulbs. A 16-hour light, 8-hour dark photoperiod was maintained with an automatic timer.

Based on the calibrated flow of dilution water the flow-splitting chambers cycled 180 times per 24 hours to provide 6.42 volume replacements per day.

Test vessels were glass battery jars having a total volume capacity of 1.6 L. Exposure solutions drained from each vessel through two 2-cm holes, approximately 15 cm from the bottom of the jars, which maintained the test solution volume at 1.4 L. The drain holes were covered with a Nitex® 40-mesh screen to prevent loss of the daphnids. Four replicates were maintained for all treatments and the controls.

During the definitive test, from day 0 to day 20, the daphnids were fed 3 times per day using 3 mL food suspension containing approximately 4 x 107 cells/mL of the unicellular green alga, Ankistrodesmus falcatus (ANK) per feeding interval and replicate, i.e., 9 mL food suspension per day and replicate.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.205 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% C.I. 0.0323-0.487 mg/l

Effect concentrations 2

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 3

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 800 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 4

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.342 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% C.I. 0.103-0.631 mg/l

Effect concentrations 5

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.334 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 6

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 7

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: length

Effect concentrations 8

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.151 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: length

Effect concentrations 9

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.334 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: length

Details on results:

Analyses were performed on days 0, 11, 14, 18 and 21. Concentrations measured in the exposure solutions were generally consistent yielding in percent recoveries of 38.9 to 138% of the nominal levels. The time weighted mean concentrations were 0.017, 0.057, 0.151, 0.334 and 0.800 mg test item/L.
Analysis of the quality control (QC) samples resulted in measured concentrations ranging from 83.6 to 110% (N = 14) of nominal concentrations. Based on these results, it was demonstrated that satisfactory precision and quality control were maintained during the analysis of exposure solutions.

Reported statistics and error estimates:

With the exception of survival data, male daphnids were excluded from statistical analysis.

A t-test (Sokal and Rohlf, 1981) was used to compare the performance of the dilution water
control organisms with that of the solvent control organisms. Analyses of survival, reproduction and growth (total body length) using the t-test established no significant difference between the dilution water control and solvent control. Statistical comparisons to determine treatment effects were performed utilizing dilution water control data.

The data for survival, reproduction and growth (total body length) were first checked for normality using Shapiro-Wilks' Test (Weber et al, 1989) and for homogeneity of variance using Bartlett's Test (Horning and Weber, 1985). The data set for survival was arc-sine transformed to pass the test for normality. Due to zero variance in one of the test groups, the homogeneity test could not be performed and further transformation of the data was useless.
ANOVA and Bonferroni t-Test were considered to be the most conservative test to determine the NOEC and LOEC values for survival. The data sets for reproduction and growth (total body length) passed the tests for homogeneity and normality, Bonferroni t-Test (Weber et al, 1989) was used to determine the NOEC and the LOEC.

All statistical analyses were conducted at the 95% level of certainty except in the case of the Shapiro-Wilks' Test and Bartlett's Test, in which the 99% level of certainty was applied. The 99% level of certainty is preferred for these qualifying tests. TOXSTAT® version 3.5 (Gulley et al, 1996) was used to perform the statistical calculations.

The 21-day EC10, 50 values for survival, reproduction and length were calculated based on time weighted mean measured concentrations by using bootstrap analysis.

Any other information on results incl. tables

Although male organisms became apparent during the course of the study, this is not considered to be an indication of a general problem with, or stress in, the culture used to initiate the test as no males were found in the culture itself. Two days prior to the initiation of

the test, a temperature of 16°C was recorded in the culture. This was only recorded on one day but may have been sufficient to result in the generation of males in the culture vessels from which daphnia were isolated to initiate the test. Nevertheless, the culture is considered

healthy and suitable for testing as the following criteria were consistently met during the month prior to test initiation:

• No ephippia were present in the culture

• No males were apparent in the culture

• The average time to the production of first brood was less than 12 days

• The average production of young per adult was greater than three in the seven days prior to test initiation

• No mortality was observed

and as such is not considered to have an impact on the quality of the data produced using these organisms.

First brood release among daphnids exposed to the 0.017, 0.057, 0.151, 0.334 and 0.800 mg test item/L treatment levels was observed between test days 7 and 8, which was consistent with the control and solvent control performance.

Daphnids exposed to the 0.017, 0.057, 0.151, 0.334 and 0.800 mg test item/L treatment levels released a mean number of cumulative live offspring per female of 163, 141, 170, 129 and 133, respectively.

After 21 days of exposure, the mean total body length among female daphnids exposed to the control, solvent control and the 0.017, 0.057, 0.151, 0.334 and 0.800 mg test item/L treatment levels averaged 5.04, 5.00, 4.88, 4.70, 4.89, 4.70 and 4.64 mm, respectively.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Although males were present in the test the results can still be used for classification and risk assessment as the females present performed well and if this fact would have an impact on the results this is negative (the animals will have a worse condition and will therefore be mroe sensitive) and the endpoint are lower. Therefore this study can be seen as a worst case.

Based on the most sensitive parameter (mean number of cumulative live offspring per female), the EC10 and EC50 values for Perkalink 900 and Daphnia magna were calculated to be 0.205 mg test item/L and > 0.800 mg test item/L, respectively. The 21 day NOEC and LOEC values for reproduction were determined to be 0.800 and > 0.800 mg test item/L, respectively.

Executive summary:

STUDY TITLE: Perkalink 900: Chronic reproduction test with daphnids (Daphnia magna) under flow-through conditions

AMENDMENT(S):

#1, dated 14 June 2010

#2, dated 21 June 2010

#3, dated 02 August 2010

#4, dated 25 October 2010

#5, dated 10 November 2010

SMITHERS VISCIENT AG STUDY #: 1148.000.231

TEST ITEM: Perkalink 900, Lot No.: 812191, reported to have a purity of 92.3%, was received from Flexsys, Louvain-la-

Neuve, Belgium, on 15. February 2010

TEST DATES: 10 September to 01 October 2010 (including total body length determination)

Last analytical work was performed on 03 October 2010.

TEST ORGANISMS: Daphnia magna

Age: < 24 hours

Source: Springborn Smithers Laboratories (Europe) (new Smithers Viscient AG) culture

DILUTION WATER:

Elendt M4 medium

pH: 6.74 to 7.47

Specific conductivity: 315 to 390 μS/cm

Total hardness as CaCO3: 146 to 152 mg/L

Total alkalinity as CaCO3: 27 to 31 mg/L

TEST CONDITIONS:

21-day duration, continuously measured temperature of 18 to 22°C (single point measured temperature 20 to 23°C), photoperiod of 16 hours light and .8 hours darkness.

NOMINAL TEST CONCENTRATIONS:

Control, solvent control and 0.026, 0.064, 0.16, 0.4 and 1.0 mg test item/L.

TIME-WEIGHTED MEAN MEASURED CONCENTRATIONS:

Control, and solvent control and 0.017, 0.057, 0.151, 0.334 and 0.800 mg test item/L.

RESULTS:

Offspring per female Daphnia was the most sensitive endpoint based on EC10 values. The EC10 for this endpoint was 0.205 mg/L

with 95% C.I. of 0.0323 - 0.487 mg/L.