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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). The study, if used in support of terephthalic acid, has a reliability of 1 (reliable without restriction). Proprietary GLP guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect, at the time the study was conducted.
GLP compliance:
yes
Vehicle:
no
Details on test solutions:
The test substance and the reference were tested at different concentrations in the test solution containing 16 mL of the synthetic sewage feed, 200 mL of the inoculum, and deionised water to give 500 mL final volume.
In the range finding test, special measures were introduced to achieve a correct concentration of the poorly water soluble test material within the test solution at the low concentrations. For this purpose 100.00 mg of the test material were weighed into a measuring flask and dissolved in ethylacetate. 50, 5, and 0.5 mL of this solution were transferred into the incubation flasks. The solvent was evaporated. By this procedure the test material was distributed in the incubation flask as a very thin layer. The test solutions were then prepared like the blank by adding 284 mL deionised water and 16 mL of the synthetic sewage. The test was then started by addition of 200 mL activated sludge.
Test organisms (species):
activated sludge
Details on inoculum:
Activated sludge from the sewage plant at Frankfurt a.M./Niederrad dealing predominantly with domestic sewage was washed twice with tap water. Dry weight was determined to be 14.49 g/L in the range-finding test, and 8.88 g/L in the definitive test. The activated sludge was diluted with tap water resulting in 4.0 g Dry mass/L. To this sludge suspension 50 mL of synthetic sewage was added per litre of activated sludge and the mixture was aerated for two days at room temperature until use in the test as inoculum. Each day it was fed with synthetic sewage concentrate.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
10 minutes
Hardness:
Not Reported
Test temperature:
Not Reported
pH:
Not Reported
Dissolved oxygen:
Not Reported
Salinity:
Not Reported
Nominal and measured concentrations:
Test Substance
Range-finding test: 0.001, 0.01 and 0.1 g/L
Definitive test: 0, 0.5, 1.00, 2.00 and 4.00 g/L

Reference Substance
Range-finding test: 7.50, 15.00, and 30.00 mg/L
Definitive test: 7.0, 14.0, 30.0, and 60.0 mg/L
Details on test conditions:
1 L glass bottles previously washed carefully with methanol/HCl (90/10) served as test vessels. Synthetic sewage feed was mixed with the appropriate amount of test substance (four different concentrations within the definitive test [500, 1000, 2000, and 4000 mg/L], three concentrations in the range-finding-test [1, 10, and 100 mg/L]) and made up to 300 mL with deionised water. The test was started by adding 200 mL of the prepared activated sludge (4 g dry weight/L) to give 1.6 g dry weight/L final concentration in 500 mL final volume. Each test solution was aerated with compressed air by means of glass pipettes for exactly 3 h. At the end of the 3 h exposure period the solutions were poured into an oxygen-bottle (ORI) and oxygen consumption was recorded for 10 minutes.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol at test concentrations of 7.5, 15 and 30 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1 393 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% confidence interval: 1272-1524 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
In the range finding test there was no effect on respiration. In the definitive test, the highest concentration of 4000 mg/L resulted in 95.8% inhibition of respiration, 2000 mg/L resulted in 93.4% inhibition, 1000 mg/L resulted in 29.7% inhibition, whilst 500 mg/L did not inhibit respiration.


Results with reference substance (positive control):
In the range finding test, 85.8% inhibition was achieved at 30 mg/L. In the definitive test, 97% inhibition was achieved at 30 mg/L
Reported statistics and error estimates:
The percent inhibition was plotted against concentration (log), and an EC50 value was calculated according to Litchfield and Wilcoxon with 95% confidence limits.

Duration

Endpoint

Effect conc.

3 h

EC50

1393 mg/L

3 h

NOEC

500 mg/L

3 h

EC75

1903 mg/L

3 h

EC95

2981.7 mg/L

The results of the definitive test are presented in the table below:

Terephthalic acid nominal concentration (mg/L)

respiration rate, mg O2/L*h

respiration inhibition (%)

relative to mean control

0.0
(control, mean value)

153.6

n.a.

500

180

0*

1000

108

29.7

2000

10.2

93.4

4000

6.5

95.8

7.5 mg 3,5-DCP/L (reference inhibitor)

51.6

66.4

15.0 mg 3,5-DCP-L (reference inhibitor)

10.2

93.4

30.0 mg 3,5-DCP/L (reference inhibitor)

4.5

97.0

 * respiration was stimulated, relative to the mean control.

Validity criteria fulfilled:
yes
Conclusions:
Inhibition, relative to the mean control rate occurred in the test substance treatments ≥1000 mg/L and the 3-h EC50 was 1392.8 mg/L. The respiration of activated sludge was unaffected by 3-h exposure to terephthalic acid at nominal concentrations of up to 500 mg/L. Therefore, a NOEC of 500 mg/L has been established.
Executive summary:

The toxicity of the test substance was determined in an activated sludge respiration inhibition test according to OECD guideline 209. Activated sludge obtained from a predominantly domestic sewage plant was exposed over a period of three hours to the test substance at nominal concentrations of 500, 1000, 2000 and 4000 mg/L. A pair of test vessels was allocated to the control treatment and the test substance treatments were run singly. Inhibition, relative to the mean control rate occurred in the test substance treatments ≥1000 mg/L and the 3-h EC50 was 1393 mg/L. The respiration of activated sludge was unaffected by 3-h exposure to terephthalic acid at nominal concentrations of up to 500 mg/L. Therefore, a NOEC of 500 mg/L has been established.

Description of key information

Key value for chemical safety assessment

EC50 for microorganisms:
1 393 mg/L
EC10 or NOEC for microorganisms:
500 mg/L

Additional information

The study was conducted using terephthalic acid, the primary degradate of terephthaloyl dichloride, according to OECD 209 under GLP. Activated sludge obtained from a predominantly domestic sewage plant was exposed over a period of three hours to the test substance at nominal concentrations of 500, 1000, 2000 and 4000 mg/L. Inhibition, relative to the mean control rate, occurred in the test substance treatments >1000 mg/L and the 3-h EC50 was 1393 mg/L. The respiration of activated sludge was unaffected by 3-h exposure to terephthalic acid at nominal concentrations of up to 500 mg/L. Therefore, a NOEC of 500 mg/L has been established.