5-sulphosalicylic acid

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

Study period:

5 FEB 2020 - 6 FEB 2020

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 (Carbon and Ammonium Oxidation))

Version / remarks:

as of 22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (20 +/- 5°C)
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Stability in H2O: 48 hrs

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing 9.999 g/L in deionised water was prepared. The stock solution was used to prepare the treatments.
- Controls: 3,5-Dichlorophenol was used as positive control, tap water as negative or blank control
- Test concentration separation factor: 10
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NW-Lachen-Speyerdorf, Im Altenschemel.
- Pretreatment: Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed/L.
- Initial biomass concentration: 2.48 g suspended solids/L
- Preparation of inoculum for exposure: On the day before the experiment, the inoculum was taken from its source, washed, aerated and the dry matter was determined. Volume was adapted to the desired content of dry matter (2 g/L). The nutrient solution was thawed and the sludge was fed with 50 mL nutrient solution/L sludge. On the day of the experiment, the dry matter of the inoculum was determined once more.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

not specified

Test temperature:

18.1-20.8°C

pH:

pH 7.8-8.0 in the blank control and pH 8.0-8.1 in the positive control,
pH 7.7-8.0 in the treatments

Dissolved oxygen:

not specified

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

In the absence of analytical monitoring, only nominal concentrations are given:
1 - 10 - 100 - 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers (800-1000 mL)
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 800 - 1000 mL, filled with 500 mL test solution
- Aeration: yes, with purified air by using Pasteur pipettes
- No. of vessels per concentration (replicates): 1 replicate for concentration 1-100 mg/L, 5 replicates for the highest concentration 1000 mg/L
- No. of vessels per control (replicates): 1 replicate per concentration for the positive control, 6 replicates for the blank control
- Sludge concentration (weight of dry solids per volume): 1.24 g suspended solids/L
- Nutrients provided for bacteria: 16 mL of nutrient solution per test vessel, composition see below ('Any other information...')
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Particulate matter: TOC <0.5 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: performed in darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
After 3 hours, the content of the first vessel was poured in a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the O2-concentration over a period of max. 5 minutes. The subsequent vessels were measured likewise in 5 minutes intervals.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: yes, according to OECD TG 209 (Since no statistically significant toxic effect occurred in any concentration, further testing at higher or lower concentrations is not necessary.)

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (1,3-Dichloro-5-hydroxybenzene, C6H4Cl2O, CAS-No. 591-35-5), tested concentrations: 5 - 10 - 20 - 40 mg/L

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: none
- Adsorption (e.g. of test material to the walls of the test container): none observed
- Blank controls oxygen uptake rate: 45.46 mg/(L*h) or 36.66 mg/(g suspended solids in 1 hour)
- Coefficient of variation of oxygen uptake rate in control replicates: 4%

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: 3h-EC50 = 9.5 mg/L, lying in the recommended range of 2-25 mg/L

Reported statistics and error estimates:

Statistical Determination of NOEC: For the treatments with the nominal test item concentration of 1000 mg/L, it was tested whether the differences between treatment and blank control were significant. For this determination, the values of the O2 consumption were used. In order to select a suitable test for significance, it was checked whether equality of variance was given. As equality of variance was given, the t-test was used. With the t-test, it was checked whether the differences are significant. The difference between 1000 mg/L treatment and the blank control can be considered as not significant as the calculated t-value (0.36) lay below the tabulated t-value (2.26) and O2 consumption in the treatment was higher than in the blank control. Therefore, the nominal concentration of at least 1000 mg/L is stated as NOEC.

Validity criteria fulfilled:

yes

Remarks:

Coefficient of variation of oxygen uptake rate in blank control <30%? Yes, 4%. Blank controls oxygen uptake rate >= 20 mg per g of activated sludge per hour? Yes, 36.66 mg/(g*h). EC50 of positive control between 2-25 mg/L? Yes, 9.5 mg/L.

Conclusions:

An activated sludge respiration inhibition test according to OECD TG 209 was conducted with 5-sulfosalicylic acid. The test conditions are well documented, the validity criteria were met and the results can be considered as reliable for the assessment of the toxicity of the test substance to microorganisms.
The test was conducted by incubation of samples of activated sludge with a nutrient source to several concentrations of the test material, of the positive control substance and a blank control. The positive control was running to proof the viability of the microorganisms. Following incubation under standard test conditions for a period of 3 hrs the rate of oxygen consumption was measured in each sample. Based on the obtained results the NOEC of nominal >=1000 mg/L was determined by performing the Student's t-test. The EC50 and EC10 were considered to be >1000 mg/L according to the inhibition in the highest test concentration of nominal 1000 mg/L which was far below 50% and 10% respectively.

Executive summary:

Within the present study the OECD Activated Sludge, Respiration Inhibition Test (OECD TG 209) was evaluated as a method for assessing the potential impact of chemicals on wastewater treatment systems. Reproducibility of the test method was examined with respect to variation in oxygen consumption rates measured in the blank control under identical conditions, and the variability in EC₅₀ values estimated for the reference compound 3,5-dichlorophenol.

The test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. For the highest treatment of nominal 1000 mg/L, 5 replicates were used; for the other treatments, 1 replicate each. Because no significant inhibition was observed in the range finder (NOEC: >= 1000 mg/L), no additional definite test had to be performed.
All validity criteria were met. For the estimation of the EC₅₀ of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. An EC₅₀ of 9.5 mg/L (95 % confidence interval 5.6 – 13 mg/L) was determined lying in the recommended range of 2–25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30 % at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O₂/g activated sludge in 1 hour.
No inconsistencies in the dose-response estimation could be observed. Therefore, no further experiment was performed in order to discern between inhibition of nitrificators and inhibition of total population.
No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Description of key information

OECD 209, GLP, Activated sludge, static, 3hrs, fresh-water, 1, 10, 11, 1000 mg/L nominal: NOEC (3h) ≥ 1000 mg/L, EC10 (3h) > 1000 mg/L, EC50 (3h) > 1000 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

Within the present study the OECD Activated Sludge, Respiration Inhibition Test (OECD TG 209) was evaluated as a method for assessing the potential impact of chemicals on wastewater treatment systems. Reproducibility of the test method was examined with respect to variation in oxygen consumption rates measured in the blank control under identical conditions, and the variability in EC₅₀values estimated for the reference compound 3,5-dichlorophenol.

The test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. For the highest treatment of nominal 1000 mg/L, 5 replicates were used; for the other treatments, 1 replicate each. Because no significant inhibition was observed in the range finder (NOEC: >= 1000 mg/L), no additional definite test had to be performed.
All validity criteria were met. For the estimation of the EC₅₀of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. An EC₅₀of 9.5 mg/L (95 % confidence interval 5.6 – 13 mg/L) was determined lying in the recommended range of 2–25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30 % at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O₂/g activated sludge in 1 hour.
No inconsistencies in the dose-response estimation could be observed. Therefore, no further experiment was performed in order to discern between inhibition of nitrificators and inhibition of total population.
No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.