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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

TRIGONOX R-938 is a clear colourless liquid and was treated as 100% pure.
The test substance was hardly soluble in water and was added quantitatively to the test vessels.
A concentration range of 32 to 320 mg/I forming a geometric progression with a factor 1.8 was tested. 100 mg/I was tested in duplicate.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Test System: Micro-organisms in activated sludge
- Source: Municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands.
- Number of micro-organisms: Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.
- Preparation of the sludge: The sludge was coarsely sieved, washed and diluted with tap-water. A small amount of the sludge was weighed and dried at ca. 105°C to determine the amount of suspended solids (3.2 g/I of sludge, as used for the test). Before use the pH was checked (measured value: 7.5). The batch of sludge was used on subsequent days (maximum four days), therefore 50 ml of synthetic sewage feed was added to each litre of activated sludge at the end of each working day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 min

Test temperature:

The temperature of the sludge, stored overnight in the laboratory, was 23.4°C at the start of the day of testing. Thus, this temperature was 1.4°C above the protocolled range.
Thereafter the sludge was transferred to a climate room. The study was performed in this climate room. The temperature in this room was 19.5°C.
The deviation at the start of the day of testing was considered to have no effect on the outcome of this study.

pH:

7.9-8.1

Dissolved oxygen:

At the start of the test: 6.5 - 9.7 mg/l

Nominal and measured concentrations:

Nominal: 32, 56, 100, 180 and 320 mg/l

Details on test conditions:

TEST SYSTEM
- Contact time: 30 minutes, during which aeration and stirring took place.
- Vessels: All glass, 300 ml oxygen bottles and 1 I test bottles.
- Milli-RO / Milli-Q water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ionexchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).
- Synthetic sewage feed:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCI
0.4 g CaCI2.2H20
0.2 g MgS04 .7H20
2.8 g K2HP04
Dissolved in 1 I Milli-Q water and filtered.
The pH was 7.0.
- Air supply: Clean, oil-free air.
- Oxygen meter: Oxygen electrode (Tri Ox EO 200, WTW, FRG) supplied with a recorder (Kipp BD40).
- Performance of the test: The synthetic sewage feed (16 ml) and an adequate amount of the test substance were mixed and made up to 300 ml with Milli-RO water. Activated sludge (200 ml) was added and the mixture was aerated in a 1 I bottle during the contact time, using a pipette as an aeration device. Then a well mixed sample of the contents was poured into a 300 ml oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder, forcing the air out of the vessel. Oxygen consumption was measured and recorded for approximately 10 min. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture.
This procedure was repeated for all concentrations of the test substance. In each test series two controls without test substance were tested, one at the start and one at the end. Each batch of activated sludge was checked for sensitivity by testing the reference substance 3,5dichlorophenol.
The flasks were filled up to 500 ml final volume.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% C.I. 40-103 mg/l

Details on results:

The inhibitory effect of TRIGONOX R-938 on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 27% inhibition at the lowest concentration tested (32 mg/I) to 92% inhibition at the highest concentration tested (320 mg/I).

The EC-values were determined using linear regression (regression line: Y = 67.38 X -71.91, Y = % inhibition and X = log concentration (mg/I)).

Results with reference substance (positive control):

Results were as expected.

Reported statistics and error estimates:

The EC-values were determined using linear regression (regression line: Y = 67.38 X -71.91, Y = % inhibition and X = log concentration (mg/I)).

pH, oxygen concentration at the start of measurement and the influence of 3,5-dichlorophenol and the test substance on the oxygen consumption of microbes in activated sludge and percentage inhibition in respiration rate.

Flask	Conc. Ref./test substance (mg/l)	Oxygen conc. At the start (mg O2/l)	Oxygen consumption (mg O2/l/h)	Inhibition (%)	pH
C1	-	8.0	34	-	8.0
C2	-	6.5	37	-	8.1
R1	3.2	8.5	31	13	8.0
R2	10	9.7	17	52	8.1
R3	32	9.5	8	77	8.1
T1	32	7.8	26	27	7.9
T2	56	7.7	20	44	7.9
T3	100	8.5	11	69	8.0
T4	100	8.5	13	63	8.0
T5	180	8.7	6	83	7.9
T6	320	9.4	3	92	8.0

Validity criteria fulfilled:

yes

Conclusions:

The EC50 was 65 mg/I with a 95% confidence interval ranging from 40 to 103 mg/l.

Executive summary:

The influence of the test substance on the respiration rate of activated sludge was investigated after a contact time of 30 minutes.

The study procedure was based on OECD Guideline No. 209, adopted April 4, 1984 and EEC Directive 67/548 amended November 18, 1987 (87/302), Part C, Publication No. L133, adopted May 30, 1988.

The test substance was hardly soluble in water and was added quantitatively to the test vessels. A concentration range of 32 to 320 mg/I forming a geometric progression with a factor 1.8 was tested. 100 mg/I was tested in duplicate.

The inhibitory effect of the test substance on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 27% inhibition at the lowest concentration tested (32 mg/I) to 92% inhibition at the highest concentration tested (320 mg/I).

The EC-values were determined using linear regression (regression line: Y =67.38 X -71.91, Y = % inhibition and X = log concentration (mg/I)).

The respiration rates of the controls were within 15% of each other. The EC50 of the reference substance, 3,5-dichlorophenol, was 11 mg/l. Therefore, the test was considered to be valid.

In conclusion, under the conditions of this present test, the test substance was toxic to waste water (activated sludge) bacteria at and above 32 mg/I, the lowest concentration tested.

The EC10 was 16 mg/I with a 95% confidence interval ranging from 9 to 31 mg/l.

The EC20 was 23 mg/I with a 95% confidence interval ranging from 13 to 41 mg/l.

The EC50 was 65 mg/I with a 95% confidence interval ranging from 40 to 103 mg/l.

The EC80 was 180 mg/I with a 95% confidence interval ranging from 111 to 290 mg/l.

Description of key information

The EC10 was 16 mg/L

The EC50 was 65 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:

65 mg/L

EC10 or NOEC for microorganisms:

16 mg/L