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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1993-08-18
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP guideline study
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE CATEGORY APPROACH
The read across follows Scenario 5 - Qualitatively and quantitatively similar effects are caused by a common compound, which is formed from all category members (as described in the 2017 Read-Across Assessment Framework document).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Benzenesulfonic acid, di-C10-14-alkyl derivs., sodium salts
SOURCE: Sulfonic acid, petroleum, calcium salt
3. CATEGORY APPROACH JUSTIFICATION

Linear and non-linear or branched alkylbenzene sulfonates are anionic surfactants with molecules characterized by a hydrophobic (apolar) and a hydrophilic (polar) group. As a group of chemicals, they are generally mixtures of closely related isomers and homologues. Each molecule contains an aromatic ring sulfonated at the para position and attached to either a linear or a branched alkyl chain at any position except the terminal carbons. The sulfonate group is a common functional group present in each of the category members, and is expected to exhibit similar biological activities with little influence from the length of carbon chain. The cation components of the chemicals (e.g. calcium, magnesium, sodium, or barium) are not expected to contribute significantly to the toxicity.
4. DATA MATRIX
See Read Across document attached to CSR
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Remarks:
no details available
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
- Name of test material (as cited in study report): OS #16928AK
- Substance type: thick dark colored liquid
- Analytical purity: 100 % active ingredient
- Lot/batch No.: 130640-002
Analytical monitoring:
no
Details on sampling:
After three hours of exposure, or in the case of the controls, three hours within the test vessel, 300 mL of the sludge mixture were transferred into a BOD bottle containing a tefloncoated magnetic stir bar. The bottle was capped with the BOD bottle oxygen probe to eliminate air space and placed on a magnetic stirring plate. The sludge mixture was stirred at a constant rate and the consumption of oxygen over time was recorded on a strip chart recorder. The test material control or reference toxicant concentration was written on the strip chart paper. The oxygen readings were recorded for ten minutes or until a linear trace covering a sufficient range of oxygen concentrations was obtained.
Vehicle:
no
Details on test solutions:
REFERENCE SUBSTANCE
The standard reference toxicant for this type of sludge respiration assessment is 3,5-dichlorophenol (97 % pure; Aldrich Chemical, Milwaukee, WI; Lot #09905CZ). A stock solution of the reference toxicant was prepared following the methods outlined in OECD 209 (OECD, 1984). One-half gram of 3,5-dichlorophenol was dissolved in 10 mL of a 1N NaOH solution which was subsequently diluted to 30 mL with WCC laboratory reagent grade water, placed on a magnetic stirrer and stirred with a tefloncoated stir bar, followed by the addition of 8 mL of 1N H2S04 (ACS grade). The solution was then brought to a total volume of 1 L with WCC laboratory reagent grade water for a nominal concentration of 500 ppm.

EXPOSURE INITIATION
The sludge solution was diluted to a concentration of 4 g/L using dechlorinated water. The following sequence was followed to initiate the test. The first test vessel was used as the first control in the testing series. Sixteen mL of the synthetic sewage feed, 284 mL of dechlorinated tap water, and 200 mL of the sludge inoculum were added to this vessel. The vessel was immediately placed in the temperature-controlled incubator and aerated.
Fifteen minutes later, the reference toxicant series was begun. In a separate test vessel, sixteen mL of the sewage feed was added, followed by 274 mL of dechlorinated water, 10 mL of the reference toxicant stock solution, and 200 mL of the sludge inoculum. As with the first control, the vessel was immediately placed in the temperature-controlled incubator and aerated. This process was repeated at fifteen-minute intervals with 20 mL and 40 mL of reference toxicant solution, respectively. For each reference toxicant addition, the volume of dechlorinated water was adjusted so that the total volume within the test vessel remained at 500 mL. The nominal reference toxicant concentrations thus were 10, 20 and 40 ppm, respectively.

Once the series of three reference toxicant test vessels were started, test vessels containing the test material were prepared. As the test material was considered insoluble, no stock solution was prepared and the material was added directly to each test vessel by way of glass microscope cover slips. The nominal test material concentrations were 1, 10, 100, 1,000 and 10,000 mg/L. Thus for 1 mg/L, 0.0005 g was added to the test vessel for 10 mg/L, 0.0050 g; for 100 mg/L 0.0500 g; for 1,000 mg/L 0.5000 g; and for 10,000 mg/L 5.000g. Each vessel was set up at fifteen-minute intervals. After the last test material vessel was prepared, a vessel with no test material was prepared as a test control replicate.
Test organisms (species):
activated sludge of a predominantly industrial sewage
Details on inoculum:
Activated sludge with associated aerobic organisms were obtained from the Cottonwood Subdivision Wastewater Treatment Plant located in Franklin, Tennessee. This wastewater treatment plant serves only the residential subdivision and its community pool. One gallon of activated sludge was collected by the plant operator, picked up on the day of collection by WCC personnel and transported to the testing laboratory. Immediately upon arrival, the sludge was aerated with low-pressure, oil-free air. The activated sludge organisms were fed a synthetic sewage feed at a rate of 50 mL per liter.

Synthetic Sewage Medium (per 1 L of solution):
16.0 g Peptone (Type I, Sigma Chemical, Lot #32H0025), 11.0 g Beef Extract (Difco Lot #2900 expiration Dec, 1995), 3.0 g Urea (Sigma Chemical, Lot #71H0574), 0.70 g NaCI (99 % +, Aldrich Chemical, Lot #764714-5), 0.40 g CaC12 -2H2O (Fisher Chemical, Lot #915231), 0.20 g MgSO4- 7H2O (Sigma Chemical, Lot #108F-0255), 2.80 g K2HPO4 (Sigma Chemical, Lot #121H0637)
Triplicate 4 mL samples of the mixed sludge were dried at 100 °C in a Fisherbrand drying oven, until a constant weight was achieved. Based on these results the sludge was diluted to produce a dry weight per unit volume concentration of 4 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
No post exposure observation period reported.
Hardness:
The dilution water for this test was from the City of Franklin, Tennessee, water supply. The water was softened and dechlorinated prior to use. Dechlorination was confIrmed with the DPD colorimetric method (APHA, 1989).
Test temperature:
20 °C.
pH:
No details available.
Dissolved oxygen:
After three hours of exposure, or in the case of the controls, three hours within the test vessel, 300 mL of the sludge mixture were transferred into a BOD bottle containing a tefloncoated magnetic stir bar. The bottle was capped with the BOD bottle oxygen probe to eliminate air space and placed on a magnetic stirring plate. The sludge mixture was stirred at a constant rate and the consumption of oxygen over time was recorded on a strip chart recorder. The test material control or reference toxicant concentration was written on the strip chart paper. The oxygen readings were recorded for ten minutes or until a linear trace covering a sufficient range of oxygen concentrations was obtained.
Salinity:
Not applicable.
Nominal and measured concentrations:
1, 10, 100, 1000 and 10000 mg/L
Details on test conditions:
TESTING SYSTEM
The testing system consisted of two units:
1) a system used to expose the sludge inoculum to the test material
2) a system to measure the rate of oxygen consumption of the sludge in a closed bottle.

The sludge / microbial inoculum was exposed to the test material in 1 L glass serum bottles. All bottles and glassware used in testing were cleaned with soap, 10 % nitric acid, acetone and WCC reagent grade water. Low-pressure oil-free air was delivered to each serum bottle using a glass pipette. Air pressure was controlled by lines connected to a pressurized manifold. All of the test vessels were held in a Forma Scientific Model 3956 water-jacketed incubator in the dark during the three-hour exposure period. The incubator maintained a constant temperature of 20 °C.

To measure the rate of oxygen consumption for the activated sludge after exposure to the test materials a Model 51 Yellow Springs Instruments (YSI) dissolved oxygen meter was used. The oxygen meter was fitted with a BOD bottle probe, with a standard sensitivity membrane and an external output to a Fisher Recordal (Model 5000) strip chart recorder. The strip chart recorder was set at the 100-mV range and run at 1 cm per minute. To measure the consumption of oxygen, a sub-sample of the exposed sludge was placed in a clean BOD bottle, the change in oxygen concentration over time was measured with the YSI meter and probe, and the results were recorded on the strip chart recorder.

TEST MEDIUM / WATER PARAMETERS
The dilution water for this test was from the City of Franklin, Tennessee, water supply. The water was softened and dechlorinated prior to use. Dechlorination was confIrmed with the DPD colorimetric method (APHA, 1989).

ANALYSIS OF CONSUMPTION DATA
Only the linear section on the chart paper representing the oxygen consumption was used to evaluate the results. The chart readings were transformed into mg of O2 per liter consumed per hour (mg O2/L*h). To calculate the inhibitory effect of the test material, the respiration rate is expressed as a percentage of the two control respiration rates for each test material concentration:

1 - [(2 x Rs)/(Rc1 + Rc2)] x 100

where:
Rs = oxygen consumption rate of the inoculum exposed to the test material;
Rc1 = oxygen consumption rate, Control 1;
Rc2 = oxygen consumption rate, Control 2.

In addition to the assessment of the test material, the reference toxicant results were similarly evaluated. The reference toxicant data were subjected to probit analysis (Litchfield & Wilcoxon, 1949) to calculate the EC50. The EC50 is the concentration of reference toxicant that produces a 50 % reduction in the oxygen consumption of the inoculum. To assure quality control, the two control consumption rates need to be within 15 % and the 3-hr EC50 of the reference toxicant (3,5-dichlorophenol) needs to be between 5 and 30 mg/L. Both of these conditions were met for the test reported herein.

To calculate the No Observable Adverse Effect Concentration (NOAEC), a 'T' test was performed following the guidelines in Sokal & Rolhf (Box 9.7; 1981). The test is a comparison between a single observation and the mean of a sample. The equation for this test is:

ts = (Y1-Mean)/(S2 x square root (N2 +(1/N2))

with: N2 -1 degrees of freedom
where: ts = the test statistic
Y1 = the test material oxygen consumption rate
S2 = the standard deviation for the mean control oxygen consumption rate
N2 = the number of control observations
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
other: NOAEC
Effect conc.:
10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
CONTROL RESPIRATION RESULTS:
The fIrst control vessel showed a change of 3.7 mg/L for oxygen concentration for a 8 1/6-minute period. This equates to an oxygen consumption rate of 27.0 mg O2/L*h. The second control vessel showed a change in oxygen concentration of 2.2 mg/L for 4 3/6-minutes. The second control thus had an oxygen consumption rate of 29.0 mg O2/L*h. The average for the two controls was 28.0 mg O2/L*h.
Results with reference substance (positive control):
REFERENCE TOXICITY RESULTS
Test inoculum was exposed to three concentrations of 3,5-dichlorophenol: 40, 20, and 10ppm. The 40-ppm test concentration showed a change of 0.40 mg O2/L for 9 4/6-minutes, or a respiration rate of 2.5 mg O2/L/h. The sludge exposed to 20 ppm of the reference toxicant produced an oxygen consumption of 0.9 mg O2/L over a 8 1/6-minute period with the respiration rate being equal to 6.6 mg O2/L/h. The 10-ppm test inoculum consumed 1.6 mg O2/L over a period of 5 3/6-minutes with a respiration rate equal to 17.5 mg O2/L*h. The resultant consumption rates for the inoculum exposed to the reference toxicant expressed as a percentage of the control were used to calculate the EC50 of the reference toxicant in the sludge used to test the experimental material. Expressed as a percent inhibition compared to the control: the microbes in the 40-ppm test were 91.1 % inhibited the microbes in the 20-ppm test were 76.4 % inhibited; and the microbes in the 10-ppm test were 37.5 % inhibited. Plotting percent effect (% inhibition) as a probit value and concentration as a log (base 10) value, the EC50 was calculated to be 13.0 mg 3,5-dichlorophenol per liter.
Reported statistics and error estimates:
STATISTICAL ANALYSIS OF THE RESULTS
The resultant respiration rates were respectively; 24.0, 24.6, 25.3, 26.7, and 28.1 mg O2/L*h. These data translate into percent inhibitions of 14.3 %, 12.1 %, 9.6 %, 4.6 %, and -0.4 %. Because no test concentration produced an inhibition greater than 50 %, an EC50 was not calculated. The best estimate for the EC50 is that it exceeds 10,000 mg/L. Each respiration rate was statistically compared as a single value versus the mean of the controls following the procedures of Sokal & Rohlf (1981) using a single-sided 'T' test. The results of the 'T' test revealed that none of the concentration expressed a statistically significant inhibition of oxygen consumption as compared to the controls. The discriminatory power of the test is low, however, due to having only one degree of freedom.

Test Material Data (OS #16928AK)

Test Concentration (mg/L)

Change in O 2(mg/L)

Change in

Time (minutes)

O 2

Consumption (mg/L*h)

Percent Inhibition

1

4.4

11

24.0

14.3

10

3.6

8 5/6

24.6

12.1

100

3.8

9

25.3

9.6

1 000

3.2

7 1/6

26.7

4.6

10 000

2.2

4 4/6

28.1

-0.4

Validity criteria fulfilled:
yes
Remarks:
Scientific validity criteria met
Conclusions:
The study report describes a valid guideline study with GLP compliance The NOAEC was calculated to be equal to 10000 mg/L. The EC50 was greater than 10000 mg/L, thus the test substance can be regarded as non-toxic to activated sludge.
Executive summary:

A study has been conducted to assess the potential environmental impact and waste control for Sulfonic acids, petroleum, calcium salts (CAS 61789-86-4), whereby the testing procedure was in accordance to OECD Guideline 209 (Goodrich, 1994). The testing strategy incorporates the potential effect of new materials on aerobic microorganisms associated with the biological treatment of wastewater. The assessment was based on the potential of a material to inhibit the metabolism of these microbes, as indicated by the microbes consumption of oxygen. This test methodology is not definitive but rather a screening mechanism for the rapid assessment of a material's potential impact. As a screening tool, the assessment of the inhibitory effect on the oxygen consumption of microorganisms within a microbe population not previously exposed to industrial wastes represents a "worst case" scenario. The test duration was a three-hour exposure period to the test material followed by up to ten minutes for the measurement of oxygen consumption. The study design was comprised of five nominal exposure concentrations: 1, 10, 100, 1000 and 10000 mg/L, a duplicate control group; and an assessment of the sensitivity of the inoculum used in the test to a reference toxicant (3,5- dichlorophenol). The activated sludge respiration test passed the quality control criteria for an acceptable test. The EC50 calculated for the reference toxicant was 13.0 mg/L, within the acceptable range of 5 to 30 mg/L. The two control replicates produced oxygen consumption rates within the required 15 % of each other, 27 and 29 mg O2/L*h. The respiration rates of the sludge-associated microbes exposed to the five nominal concentrations of the test substance were 24.0, 24.6, 25.3, 26.7, and 28.1 mg O2/L*h. respectively. The calculated No Observed Adverse Effect Concentration (NOAEC) based on inhibition compared to the control was calculated to be equal to 10000 mg/L. The EC50 was > 10000 mg/L.

Description of key information

An OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test) study on the read across source substance sulfonic acids, petroleum, calcium salts (CASN 61789-86 -4)

was performed using five nominal concentrations. The calculated No Observed Adverse Effect Concentration (NOAEC) based on inhibition compared to the control was calculated to be equal to 10000 mg/L. The EC50 was > 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
10 000 mg/L
EC10 or NOEC for microorganisms:
10 000 mg/L

Additional information

A study has been conducted to assess the potential environmental impact and waste control on the read across source substance sulfonic acids, petroleum, calcium salts (CASN 61789-86-4), in accordance to OECD Guideline 209 (Goodrich, 1994). The testing strategy incorporates the potential effect of new materials on aerobic microorganisms associated with the biological treatment of wastewater. The assessment was based on the potential of a material to inhibit the metabolism of these microbes, as indicated by the microbes consumption of oxygen. The test duration was a three-hour exposure period to the test material followed by up to ten minutes for the measurement of oxygen consumption. The study design was comprised of five nominal exposure concentrations: 1, 10, 100, 1000 and 10000 mg/L, a duplicate control group; and an assessment of the sensitivity of the inoculum used in the test to a reference toxicant (3,5- dichlorophenol). The activated sludge respiration test passed the quality control criteria for an acceptable test. The EC50 calculated for the reference toxicant was 13.0 mg/L, within the acceptable range of 5 to 30 mg/L. The two control replicates produced oxygen consumption rates within the required 15% of each other, 27 and 29 mg O2/L*h. The respiration rates of the sludge-associated microbes exposed to the five nominal concentrations of the test substance were 24.0, 24.6, 25.3, 26.7, and 28.1 mg O2/L*h. respectively. The calculated No Observed Adverse Effect Concentration (NOAEC) based on inhibition compared to the control was calculated to be equal to 10000 mg/L. The EC50 was > 10000 mg/L.