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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
Study period:
02-12-2011 - 06-12-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed under GLP according guidelines, all quality criteria were met
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See Chapter 13 for more info on the Read across
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
minor deviation
Principles of method if other than guideline:
The average oxygen uptake rate for the control replicates was slightly lower than prescribed in the guideline
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts was added to the incubation
vessels using a stock solutions of approximately 1.0 g/L. The Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts stock solution was prepared by stirring 0.12 g Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts in 120 mL of deionized water. The Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts stock solution was a clear solution. pH stock solution = 6.7
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Unadapted secondary activated sludge was obtained (05-12-2011) from the WWTP Nieuwgraaf in Duiven. The WWTP Duiven is an activated sludge plant treating predominantly domestic wastewater. Prior to use the activated sludge was homogenized with a syringe. The dry weight of the homogenized activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration. The dry weight of the activated sludge in the incubation vessels was 1.5 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
Temperature varied from 20.1 to 20.9°C.
pH:
The pH of the reaction mixtures after the incubation period ranged from 6.9 to 7.1.
Dissolved oxygen:
aerobic during test
Details on test conditions:
-The test was performed in 300 mL Erlenmeyers.
-The homogenized activated sludge was incubated in a shaking water bath (100 rpm, 20°C) for 3 hours with various concentrations of the test
compound and synthetic sewage.
-Synthetic sewage was added to the activated sludge (1.6 mL on a total volume of 50 mL).
Synthetic sewage contained per liter: 16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.3 g CaCl2
0.2 g MgSO4.7H2O
2.8 g K2HPO4
- Different concentrations (spaced by a factor two) of test compound and reference compound where added using stock solutions both of 1.0 g/L
-Tap-water was used to fill up the total test volume to 50 mL.
- The dry weight concentration of the activated sludge in the test was 1.5 g/L
-The tested concentrations of the reference compound 3,5-dichlorophenol where: 1.3, 2.5 mg/L, 5.1 mg/L,10.2 mg/L, 20.3 mg/L and 40.6 mg/L
-The tested concentrations of tris (2-hydroxyethyl) coco diaminopropane where : 40 mg/L, 80 mg/L, 160 mg/L, 320 mg/L and 640 mg/L.
-Controls without test substance (or reference compound) are measured at the beginning and at the end of the test
-The inhibitory effect of the test substance/reference compound is expressed as a percentage of the mean of the two control respiration rates.
-An abiotic control was not performed because neither reduction of oxygen by the test substance nor formation of oxygen due to chemical breakdown of the test substance is expected.
-No specific analyses were performed
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
420 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
651 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: EC50 Extrapolated: with 95% confidence limits of 439 and 1221 mg/L
Details on results:
Test conditions
The pH of the reaction mixtures after the incubation period ranged from 6.8 to 7.1. Temperature varied from 20.1 to 20.9°C. These conditions allow
respiration of the activated sludge used.

Validity of the test
The validity of the test is demonstrated by the following criteria. First, the coefficient of variation of the replicates of the control oxygen uptake rates was 5%, which is smaller than the maximum prescribed variation of <30%. Secondly, the EC50 of the reference compound was 12 mg/L which is withinthe prescribed range of 2 to 25 mg/L (Table I and Figure 1). The third criterion, an oxygen uptake rate of >20 mg O2/g dry weight/hour for the control measurements, was not achieved. The inoculum used was obtained from a treatment plant which is operated at a low organic sludge load and therefore has a natural lower oxygen uptake rate. The average oxygen uptake rate for the control replicates was 18 mg O2/g dry weight/hour, which is slightly lower than the prescribed rate. Accurate oxygen uptake rate measurements were very well possible, as shown by the very low coefficient of variation between the control replicates and therefore this is a minor deviation which is considered not to invalidate the test.

Activated sludge respiration inhibition test
The calculated respiration rates and inhibition percentages of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts are presented in Table II and the results of the graphical probit analysis are shown in Figure 2. The inhibitory effect of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts at a particular concentration is expressed as a percentage of the two controls. From these results EC values were calculated. The EC50 of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts for activated sludge after 3 hours contact time is 651 mg/L with 95% confidence limits of 439 and 1221 mg/L. The EC10, EC20 and EC80 are 420, 488 and 867 mg/L, respectively. Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts is therefore considered not harmful to activated sludge.
Results with reference substance (positive control):
The EC50 of the reference compound was 12 mg/L at a contact time of 3 hours, respectively. These EC50 values is within the prescribed range of
2 to 25 mg/L.
Reported statistics and error estimates:
Normally the EC10,20,50,80 values are computed from the best fitted line (least-squares method) through the points given by the probit of the percentage of inhibition and the logarithm of the concentration of the test substance.
The Lowest Observed Effect Concentration (LOEC) was determined by comparison of the inhibition at each concentration and the control. Confidence limits were computed on the basis of Fieller’s theorem if possible. All computations were performed using the TOXCALC version 5.023 program.
Validity criteria fulfilled:
yes
Remarks:
see "details on results"
Conclusions:
The test is valid as shown by the EC50 of the reference compound, 3,5-dichlorophenol (12 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (5%). The average oxygen uptake rate for the control replicates is slightly lower (18 mg O2/g dry weight/hour) than the prescribed rate. The accuracy of the oxygen uptake rate measurements is not affected by the slightly lower oxygen uptake rates, as shown by the low coefficient of variation for the replicates of the control oxygen uptake rates, and herewith does not invalidate the test. The toxicity of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts at a particular concentration is expressed as a percentage of the two controls. The EC50 of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts for activated sludge after 3 hours contact time is 651 mg/L with 95% confidence limits of 439 and 1221 mg/L.
The EC10, EC20 and EC80 are 420, 488 and 867 mg/L, respectively. Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts is therefore considered not harmful to activated sludge.
It should be noted that the highest concentration tested was 640 mg/L and that the EC50 and EC80 are based on extrapolation.
Executive summary:

In order to predict effects of chemicals in the environment and in biological waste water treatment plants, an activated sludge respiration inhibition test was performed. The toxicity to activated sludge was determined in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice. The test is valid as shown by the EC50 of the reference compound, 3,5- dichlorophenol (12 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (5%). The average oxygen uptake rate for the control replicates is slightly lower (18 mg O2/g dry weight/hour) than the prescribed rate. The accuracy of the oxygen uptake rate measurements is not affected by the slightly lower oxygen uptake rates, as shown by the low coefficient of variation for the replicates of the control oxygen uptake rates, and herewith does not invalidate the test. The toxicity of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts at a particular concentration is expressed as a percentage of the two controls. The EC50 of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts for activated sludge after 3 hours contact time is 651 mg/L with 95% confidence limits of 439 and 1221 mg/L. The EC10, EC20 and EC80 are 420, 488 and 867 mg/L, respectively. Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts is therefore considered not harmful to activated sludge.

Description of key information

The toxicity to microorganisms by Sodium diisobutylnaphthalenesulphonate is read across from the structural analogue Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts (CAS no 1258274 -08 -6). For algae and daphnia it has been shown that, similar to most other chemicals, longer alkyl chain naphthalene sulphonates are more ecotoxic than shorter alkyl chain naphthalene sulphonates. Read across from the source chemical, Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts (CAS no 1258274 -08 -6) to the structural analogue (target chemical) Sodium diisobutylnaphthalenesulphonate is therefore considered justified.

The toxicity of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts (CAS no 1258274 -08 -6) to microorganisms was evaluated in the respiration inhibition test according to an OECD 209 test under GLP conditions. The observed effects were 3h-EC10 for this substance for activated sludge was 222 mg/L and the 3h-EC50 was 345 mg/L (concentration based on active ingredient).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
222 mg/L

Additional information

The toxicity to microorganisms by Sodium diisobutylnaphthalenesulphonate is read across from the structural analogue Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts (CAS no 1258274 -08 -6).

The toxicity of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts (CAS no 1258274 -08 -6) to activated sludge was determined in a respiration inhibition test in accordance with OECD Test Guideline 209, and in compliance with GLP. The toxicity of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The EC50 was reported as 651 mg/L with 95% confidence limits of 439 and 1221 mg/L based on testmaterial. The EC10 was 420 mg/L based on test material. The EC50 is 345 mg/L and EC10 is 222 mg/L based on active ingredient concentration. The substance is therefore considered not harmful to activated sludge (Reliability 1).

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