dimethyl 1,2-benzenedicarboxylate;reaction mass of: 1,2-dimethylpropylidene dihydroperoxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to protocol. Not all details on chemical analysis are given but enough to validate the study.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

During the final test samples for analysis were taken in duplicate from all test concentrations and the blank-control.
- Frequency: at t=0 h, t=24 h and t=72 h
- Volume: 6ml
- Storage: Samples were stored in a deep-freeze until analysis.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, reserve samples of 12 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for possible analysis.

Vehicle:

no

Details on test solutions:

Preparation of test solutions started with stock solutions at nominally 100 mg/I (range-finding test) or 10 mg/I (final test). These solutions were magnetically stirred for ca. 20 minutes and additionally treated with ultrasonic waves for 5 minutes during the range-finding test. The resulting, clear and colourless, stock solutions were then used to prepare the lower test concentrations by subsequent dilutions in test medium. After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, adequate volumes of an algal suspension were added to each replicate providing a cell density of 10^4 cells/ml.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Species: Selenastrum capricornutum, strain: NIVA CHL 1.
- Control of sensitivity: The results of the most recent reference test with potassium dichromate (Merck, Art. 4864) are appended to this report.
- Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (4000-9000 lux) in a climate room at a temperature of 23 ± 2°C.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2.10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/l as CaCO3

Test temperature:

23.8-24.7 °C

pH:

8.3-9.3

Nominal and measured concentrations:

Nominal: 0.46, 1.0, 2.2, 4.6, 10 mg/l
Measured: n.m., 0.2, 0.5, 1.2, 6.0 mg/l
n.m: Not measured

Note from the reviewer: The lowest test concentration was not measured and the second and third lowest were on average below the detection limit. As effects on growth rate were only significant at the second highest test concentration this is not considered to have an impact on the outcome of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass
- Material, size, headspace, fill volume: 50 ml
- Aeration: no
- Initial cells density: 1*10^4 cells/ml
- Control end cells density: 127.5*10^4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per highest concentration without algae: 2
- No. of vessels per concentration with algae for chemical analysis after 24h: 1

GROWTH MEDIUM
- Standard medium used: yes, M2 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 medium
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 70-98 µE/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber at the beginning of the test; spectrophotometer at 720 nm for all other measurements

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations: 0.1, 1, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: slight inhibition at 1 mg/l and 100% inhibition at 10 and 100 mg/l.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.61 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I. 0.42-0.90 mg/l

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I. 1.2-2.5 mg/l

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% C.I. 0.08-0.93 mg/l

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.98 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% C.I. 0.30-3.2 mg/l

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

CHEMICAL ANALYSIS
It was known that the analytical method was not sensitive enough to measure the nominal concentration of 0.46 mg/1. Consequently, this concentration was not measured. The results of analysis also indicated that the measured concentration at nominally 1.0 mg/l was below the limit of detection at all measuring times. At 2.2 and 4.6 mg/I the measured concentration decreased below the detection limit after 72 hours of exposure. In conclusion: actual concentrations at the lower test concentrations were difficult to obtain.
The actual range of TRIGONOX R-938 concentrations tested was based on the average exposure concentrations of both main components. As the TWA concentrations of the components did only marginally differ, it was acceptable to base the actual test range on the mean of both TWA concentrations. The actual test concentrations were 0.2, 0.5, 1.2 and 6.0 mg/l at nominally 1.0, 2.2, 4.6 and 10 mg/l, respectively.

Results with reference substance (positive control):

Results as expected

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline (201, adopted 7 June 1984) were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth or inhibition of growth rate (ANOVA, Tukey test, Bonferroni t-test, TOXSTAT Release 3.5,1996, D.o. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of the EC50 and EC10 values was based on linear regression analysis of the percentages of growth inhibition and the percentages of growth rate reduction versus the logarithms of the corresponding TWA exposure concentrations of the test substance.

Measured concentrations versus nominal concentrations

Nominal conc. (mg/l)	Measured conc. (mg/l) Peak 1 / Peak 2			Time Weight Average (mg/l) Peak 1 / Peak 2 / Mean
	T=0h	T=24h	T=72h
1.0	0.32^1/ 0.32^1	0.32^1/ 0.32^1	0.064^2/ 0.063^2	0.20 / 0.20  0.2
2.2	1.45 / 1.33	0.70 / 0.73	0.064^2/ 0.063^2	0.48 / 0.47  0.5
4.6	3.27 / 3.08	2.55 / 2.40	0.064^2/ 0.063^2	1.23 / 1.2    1.2
10	8.05 / 7.66	7.42 / 6.67	4.39 / 3.47	6.38 / 5.59  6.0
10 (w.a.)	8.45 / 8.00	7.98 / 7.36	6.42 / 5.51	7.51 / 6.80  7.2

w.a.: without algae

¹The final exposure concentration was taken as half the lower limit of quantification

²The final exposure concentration was taken as a factor of 10 below the limit of detection as the substance was not detected

Cell densities calculated from the individual extinction values (*10^4 cells/ml)

Nominal conc. (mg/l)	Vessel no.	Exposure time (h)
		0	24	48	72
Control	1 2 3 4 5 6	1.00 1.00 1.00 1.00 1.00 1.00	2.92 3.59 3.17 3.55 4.08 4.46	32.62 34.11 32.78 26.40 33.11 32.41	130.38 147.40 125.21 119.00 120.57 122.56
0.46	1 2 3	1.00 1.00 1.00	5.04 5.57 5.95	24.79 28.39 34.40	132.33 155.06 133.08
1.0	1 2 3	1.00 1.00 1.00	5.16 5.41 6.11	30.92 27.23 35.80	121.52 109.97 166.58
2.2	1 2 3	1.00 1.00 1.00	4.41 4.04 4.79	21.89 22.51 25.70	93.53 100.94 109.14
4.6	1 2 3	1.00 1.00 1.00	3.38 3.67 4.29	7.31 6.53 10.42	24.21 19.82 42.59
10	1 2 3	1.00 1.00 1.00	1.47 1.39 1.31	2.47 1.47 1.56	1.00 1.00 1.00

Validity criteria fulfilled:

yes

Conclusions:

The EC50 for growth rate reduction (ErC50: 0-72h) was 1.7 mg/I with a 95% confidence interval ranging from 1.2 to 2.5 mg/l.
The EC10 for growth rate reduction (ErC10: 0-72h) was 0.61 mg/I with a 95% confidence intervalranging from 0.42 to 0.90 mg/l.

Based on this study TRIGONOX R-938 is classified as toxic to aquatic organisms.

Executive summary:

Selenastrum capricornutum, Fresh Water Algal Growth Inhibition Test.

The study procedures described in this report were based on the EEC Directive 92/69, Publication No. L383 Part C-3 adopted December, 1992; OECD guideline No. 201, Adopted June 7, 1984; and ISO Standard 8692, First edition, 15 November 1989.

The test substance was completely miscible with test medium at the concentrations tested.

The project started with a range-finding test exposing exponentially growing algal cultures to nominal concentrations of 0.1, 1.0, 10 and 100 mg/l. The results showed that the EC50 for cell growth inhibition and growth rate reduction was between nominally 1 and 10 mg/l.

The range-finding test was followed by a final EC50 test exposing exponentially growing algal cultures to nominal concentrations of 0.46, 1.0, 2.2, 4.6 and 10 mg/l. The study further included a blank-control. The initial cell density was 10^4 cells/ml. The total test period was 72 hours. Samples for determination of actual exposure concentrations were taken from all concentrations at the start, after 24 hours and at the end of the test period.

Analysis of the samples taken during the final test showed that concentrations decreased by more than 20% during the test period. Consequently, Time Weight Average (TWA) exposure concentrations were calculated based on both main components measured.

As the TWA concentrations of the components did only marginally differ, it was acceptable to base the actual test range on the mean of both TWA concentrations. The actual test concentrations were 0.2, 0.5, 1.2 and 6.0 mg/I at nominally 1.0, 2.2, 4.6 and 10 mg/I, respectively.

It was known that the analytical method was not sensitive enough to measure the nominal concentration of 0.46 mg/l. Consequently, this concentration was not measured.

In the controls, cell density increased by an average factor of > 16 within 3 days. Further all test conditions (pH and temperature) remained within the ranges prescribed by the protocol.

The test substance reduced growth rate of this fresh water algae species significantly at nominally 4.6 mg/I and higher, corresponding to a TWA concentration of 1.2 mg/l.

Toxicity parameters based on TWA exposure concentrations were as follows:

The EC50 for cell growth inhibition (EbC50: 0-72h) was 0.98 mg/I with a 95% confidence interval ranging from 0.30 to 3.2 mg/l.

The EC10 for cell growth inhibition (EbC10: 0-72h) was 0.28 mg/I with a 95% confidence interval ranging from 0.08 to 0.93 mg/l.

The EC50 for growth rate reduction (ErC50: 0-72h) was 1.7 mg/I with a 95% confidence interval ranging from 1.2 to 2.5 mg/l.

The EC10 for growth rate reduction (ErC10: 0-72h) was 0.61 mg/I with a 95% confidence intervalranging from 0.42 to 0.90 mg/l.

The NOEC for cell growth inhibition based on nominal concentrations was 1.0 mg/I, corresponding to a TWA concentration of 0.2 mg/l.

The NOEC for growth rate reduction based on nominal concentrations was 2.2 mg/I,corresponding to a TWA concentration of 0.5 mg/l.

Description of key information

The EC50 for growth rate reduction (ErC50: 0-72h) was 1.7 mg/I with a 95% confidence interval ranging from 1.2 to 2.5 mg/l.

The EC10 for growth rate reduction (ErC10: 0-72h) was 0.61 mg/I with a 95% confidence intervalranging from 0.42 to 0.90 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.7 mg/L

EC10 or NOEC for freshwater algae:

0.61 mg/L