Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

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:

18th November 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study to examine the effect of the test material on the respiration of activated sludge (Expanded Range Assessment), was conducted in compliance with the OECD Code of Good Laboratory Practices with the following exceptions: 1) The stability, characterization and verification of the test substance identity and, properties, the determination of the concentrations of the test substance in the test solutions by chemical analysis, and the maintenance of records for the above, concerns are the responsibility of the sponsor.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

The stability, characterization and verification of the test substance identity and, properties, the determination of the concentrations of the test substance in the test solutions by chemical analysis, and the maintenance of records for the above,

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

- Concentrations:
1.0, 10, 100, 1000 and 10 000 mg/l

- Sampling method:
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.

- Sample storage conditions before analysis:
not specified in report

Vehicle:

yes

Details on test solutions:

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 teflon-coated stir bar; followed by the addition of 8 mL of 1N H2SO4 (ACS grade). The solution was then brought to a total volume of 1L with WCC laboratory reagent grade water for a nominal concentration of 500 ppm.

- Method:
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 5.000 g. 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.

- Eluate:
The sludge/microbial inoculum was exposed to the test material in 1-L flint glass 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 test vessel using a glass pipet. Air pressure was controlled by brass needle valves. All of the test vessels were held in a Forma Scientific Mopel 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 Recprdal (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.

- Differential loading:
Not applicable.

- Controls:
The control group was maintained under identical conditions but not exposed to the test material.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable.

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
Not applicable.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
not specified

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge with associated aerobic organisms were obtained from the Cotton wood 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 (see below) 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 NaCl (99%+, Aldrich Chemical, Lot #764714-5)
• 0.40 g CaCl2 -2H20 (fisher Chemical, Lot #915231)
• 0.20 g MgSO4- 7H20 (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 degrees C in a Fisherbrand drying oven, until a constant weight was achieved (see Table 2-1 attached). 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:

Not applicable.

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 PPD colorimetric method (APHA, 1989).

Test temperature:

The test vessels were held in a Forma Scientific Model 3956 water-jacketed incubator in the dark. The incubator maintained a constant temperature of 20 degrees C.

pH:

no data supplied

Dissolved oxygen:

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:

1.0, 10, 100, 1000 and 10,000 mg/l

Details on test conditions:

EFFECT PARAMETERS MEASURED

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 litre consumed per hour (mg O2/L*hr). 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 R2)/(Rcl + Rc2)] x 100

where:

Rs = oxygen consumption rate of the inoculum exposed to the test material;
Rcl = oxygen consumption rate, Control 1; and
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.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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

Remarks on result:

other: CL not specified

Details on results:

CONTROL RESPIRATION RESULTS
The raw data collected for the control respiration results are attached in Appendix C . The fIrst control vessel showed a change of 6.5 mg/L for oxygen concentration for a 7-minute period. This equates to an oxygen consumption rate of 55.7 mg O2/L*hr. The second control vessel showed a change in oxygen concentration of 3.3 mg/I for 3 4/6-minutes. The second control thus had an oxygen consumption rate of 54.0 mg O2/L*hr. The average for the two controls was 54.9 mg O2/L*hr.

REFERENCE TOXICITY RESULTS
Test inoculum was exposed to three concentrations of 3,5-dichlorophenol: 40, 20, and 10 ppm. The 40-ppm test concentration showed a change of 0.40 mg O2/L for 4 5/6-minutes, or a respiration rate of 4.9 mg O2/L*hr. The sludge exposed to 20 ppm of the reference toxicant produced an oxygen consumption of 1.7 mg O2/L over a 7-minute period with the respiration rate being equal to 14.6 mg O2/L*hr. The 10 ppm test inoculum consumed 3.2 mg O2/L over a period of 5 minutes with a respiration rate equal to 38.4 mg O2/L*hr.

The resultant consumption rates for the inoculum exposed to the reference toxicant expressed as a percentage of the control were used to calculate theEC50 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 73.4% inhibited; and the microbes in the 10 ppm test were 30.0% inhibited. Plotting percent effect (% inhibition) as a probit value and concentration as a log (base 10) value, the EC50 was calculated to be 14.2 mg 3,5-dichlorophenol per litre. The analysis for the EC50 is attached in Appendix D.

TEST MATERIAL RESULTS
Five nominal concentrations of the test material were tested with the activated sludge inoculum; 1, 10, 100, 1,000, and 10,000 mg/L. The results observed during the test are attached in the results table.

The results produced in this assessment of the test material did not reveal a significant adverse effect of the test material on the oxygen consumption of the microbes associated with activated sludge. The EC50 for this material regarding the inhibition of metabolism, as represented by respiration, was greater than 10,000 mg/L.

The test is limited in that it represents a screening test of the potential effect of the test material on microbial metabolism.

Results with reference substance (positive control):

Results with reference substance valid?
Yes.

Relevant effect levels:
The reference material gave a 3-Hour EC50 value of 14.2 mg/l, within the acceptable range 5 - 30 mg/l.

Reported statistics and error estimates:

The resultant respiration rates were respectively; 51.3, 56.0, 46.6, 57.0, and 29.3 mg O2/L*hr. These data translate into percent inhibitions of 6.5, -2.1, 15.0, -3.9, and 46.6%. Although the 10,000 mg/L test concentration did produce an inhibition greater than 25 %, none of the test concentrations produced an inhibition greater than 50 %. Therefore, an EC50 was greater than 10,000 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The results produced in this assessment of the test material did not reveal a significant adverse effect of the test material on the oxygen consumption of the microbes associated with activated sludge. The EC50 for this material regarding the inhibition of metabolism, as represented by respiration, was greater than 10,000 mg/L. The test is limited in that it represents a screening test of the potential effect of the test material on microbial metabolism.

Executive summary:

The potential impact of the test material on microbial metabolism, as represented by the consumption of oxygen, was investigated using the "Activated Sludge Respiration Inhibition Test" as prescribed by OECD (1984) and detailed in WCC Protocol OECD209 (Expanded Range Procedures), see Appendix A attached . The test was performed on November 18, 1993. 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 1,000 and 10,000 ppm; 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 with the test material passed the quality control criteria for an acceptable test. The EC50 calculated for the reference toxicant was 14.2 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; 55.7 and 54.0 mg O2/L*hr.

The respiration, rates of the sludge-associated microbes exposed to the five nominal concentrations of the test material were 51.3, 56.0, 46.6, 57.0, and 29.3 mg O2/L*hr respectively. The EC50 was greater than:10,000 mg/L.

Description of key information

3h EC50 = >10,000 mg/L; study performed in line with OECD Guideline 209; Goodrich (1994)

Additional information

In a GLP compliant activated sludge respiration inhibition study conducted in line with standardised guidelines, the effect of the test substance on microbial metabolism, as represented by the consumption of oxygen, was investigated. The test duration was a three-hour exposure period to the test substance, followed by up to ten minutes for the measurement of oxygen consumption, at five nominal exposure concentrations: 1, 10, 100 1,000 and 10,000 ppm with a duplicate control group. The two control replicates produced oxygen consumption rates within the required 15% of each other; 55.7 and 54.0 mgO₂/L/hour. The respiration, rates of the sludge-associated microbes exposed to the five nominal concentrations of the test substance were 51.3, 56.0, 46.6, 57.0, and 29.3 mgO₂/L/hour respectively. The EC50 was determined to be >10,000 mg/L.