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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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 isophthalic acid, has a reliability of 1 (reliable without restriction).
Justification for type of information:
The test substance rapidly hydrolyses to isophthalic acid (IPA). Therefore, the study with IPA is being used to fulfil this data requirement. Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.
Reason / purpose:
read-across: supporting information
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
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 of the activated sludge was determined to be 14.49 g/L in the range-finding test, and 11.77 g/L in the main test. The activated sludge was diluted with tap water resulting 4.0 g Dry mass/L. To this sludge suspension 50 mL of synthetic sewage was added per liter 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 accordingto the OECD Guideline.

A synthetic sewage feed was made by dissolving the following amounts of substances in 1 L of aqua demin: 16 g peptone, 11 g meat extract, 3 g urea,0.7 g NaCl, 0.4 g CaCl2 x 2 H20, 0.2 g MgS04 x 7 H20, 2.8 g K2HP04.
Nominal and measured concentrations:
Test Substance
Range-finding test: 0.001 g/L, 0.01 g/L, and 0.1 g/L
Definitive test: 0.5 g/L, 1.0 g/L, 2.0 g/L, 4.0 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:
The test substance and the reference were tested at different concentrations in the test solution containing 16 mL of the "sewage feed", 200 mL of the inoculum, and deionized water to give 500 mL final volume. 1 L glass bottles previously washed carefully with methanol/HCl (90/10) served as test vessels. The test solutions were prepared according to the guideline: 16 mL of synthetic" sewage feed was mixed wIth the appropriate amount of test substance and made up to 300 mL with deionized 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. For this purpose each 15 minutes one new test solution was started. After the "contact time" of 3 h the test solutions were poured into an oxygen-bottle. (ORI), and oxygen consumption was recorded for 10 min. in order to determine the respiration rates.

For 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 ethanol. 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 deionized water and 16 mL of the synthetic sewage. The test was then started by addition of 200 mL activated sludge.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
617.1 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: 525.2 - 725 mg/L
Details on results:
EC5: 158.3 mg/L
EC25: 353.3 mg/L
EC50: 617.1 mg /L (95 % Confidence-Interval: 525.2 - 725 mg/L)
EC75: 1077.9 mg/L
EC95: 2405.4 mg/L
Results with reference substance (positive control):
EC50 = 11.0 mg/L.
The EC50 is within the range of 5 - 30 mg/L and the control values are within 15%. Therefore, the test is valid.
Reported statistics and error estimates:
The percent inhibition was calculated at each concentration. The percent inhibItion was plotted against concentration (log concentration), and an EC50 value was calculated according to Litchfield and Wilcoxon by standard procedure.

Table 1: Respiration Rates Range Finding Test

Test Substance

Reference Substance

Concentration (mg/L)

O2-Consumption

(mg O2/Lxh)

% Inhibition

Concentration (mg/L)

O2-Consumption

(mg O2/Lxh)

% Inhibition

100

64.8

activation

30.0

7.86

85.4

10

64.8

activation

15.0

11.76

78.2

1

54.6

activation

7.5

33.6

37.80

0.0

54.0

0.0

 

 

 

Table 2: Respiration Rates Definitive Test

Test Substance

Reference Substance

Concentration (mg/L)

O2-Consumption

(mg O2/Lxh)

% Inhibition

Concentration (mg/L)

O2-Consumption

(mg O2/Lxh)

% Inhibition

500

135

47.7

7.0

198

23.3

1000

79.8

69.1

14.0

76.8

70.2

2000

42

83.7

30.0

26.4

89.8

4000

2

100

60.0

10.2

96

0.0

mean 258

0.0

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).

The EC50 was calculated to be 617.1 mg/L (95% Confidence-Interval: 525.2-725 mg/L).
Executive summary:

The test substance was tested for toxicity towards microorganisms according to OECD-Test Guideline 209: "Activated Sludge, Respiration Inhibition Test". From the data obtained, the respiration rate of activated sludge from the sewage plant at Frankfurt a.M./Niederrad was not inhibited at concentrations in the range of water saturation. At concentrations higher than 100% water solubility of the test material (130 mg/L), bacteria were inhibited beginning at 158.3 mg/L (EC5). The EC50 was calculated to be 617.1 mg/L. This inhibition may be due to pH shift in the solution (after hydrolysis of the molecule). Within that same series the positive control substance 3,5-dichlorphenol, exhibited an EC50 of 11.0 mg/L.

Description of key information

The study was conducted using isophthalic acid, the primary degradate of isophthaloyl 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. The respiration rate was not inhibited at concentrations in the range of water saturation. At concentrations higher than 100% water solubility of the test material (130 mg/L), bacteria were inhibited beginning at 158.3 mg/L (EC5). The 3-hour EC50was determined to be 617.1 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
617.1 mg/L

Additional information