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

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 01 October 1997 and 14 January 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM Standard Test Method D 5864-95

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 796.3260 (Ready Biodegradability: Modified Sturm Test)

Deviations:

no

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

other: activated sludge supernatant and soil filtrate (adapted)

Details on inoculum:

Test Inoculum
Soil was collected from a wooded lot adjacent to the Wildlife facility on October 6, 1997. The soil was collected by clearing the soil surface of litter and collecting the exposed soil to a depth of approximately 20 cm. The soil was screened through a sieve with 2 mm openings. Prior to use, approximately 200 g (wet weight) of soil was suspended in 2 litres of water. The suspension was allowed to settle for approximately30 minutes and then the supernatant was filtered through glass wool. The filtrate was aerated until used.

Activated sludge was collected from Prospect Bay Waste water Treatment Facility, Grasonville, Maryland on October 8, 1997. The sludge. was sieved through a 2 mm screen and then aerated for approximately 4 hours. After the aeration period, the sludge was homogenised in a blender at medium speed for approximately 2 minutes. The sludge was allowed to settle for approximately 30 minutes. The supernatant above the settled solids was removed. The inoculum used in this study was composed of six different adaptation cultures. The cultures were maintained concurrently and were prepared as follows. Equal volumes of activated sludge supernatant and soil filtrate were combined and supplemented with 25 mg/L vitamin-free casamino acids and 25 mg/L yeast extract. A standard plate count was performed on the inoculum prior to the start of adaptation. Plates were incubated at 20 ± 3°C for approximately 48 hours. Approximately 100 mL of supplemented inoculum was combined with approximately 900 mL of test medium within each 2-L Erlenmeyer flask. The solutions were continuously aerated with CO2 :free air and the test substances were incrementally added at concentrations equivalent to 4,8, and 8 mg CIL on days 0, 7, and 11, respectively. On day 14, an equal volume of each culture was combined and the composite inoculum was screened using glass wool and then homogenised in a blender at medium speed for approximately 2 minutes. A standard plate count was performed on the inoculum. Plates were incubated at 20 ± 3°C for approximately 48 hours.

Duration of test (contact time):

28 d

Initial conc.:

10 other: mg C/l

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

INTRODUCTION
This test method was designed to measure the degree of aerobic aquatic biodegradation of a lubricant or components of a lubricant by measuring evolved carbon dioxide (CO2) upon exposure of the test material to· an inoculum. Test materials that achieve a high degree of biodegradation in this test may be· assumed to easily biodegrade in many aerobic aquatic environments. A low yield of CO2 does not necessarily mean that the test substance will not biodegrade under relevant environmental conditions but that additional testing may be needed to establish biodegradability.

This study was conducted by Wildlife International Ltd at the Wildlife International Ltd. biodegradation facility in Easton. Maryland. A copy of the raw data generated by Wildlife International Ltd. and a copy of the final report are filed under Project Number 33 lE-I 05 in the archives located on the Wildlife International Ltd. site.

OBJECTIVE
The objective of the study was to measure the amount of carbon dioxide (COJ produced from the biodegradation of the test substance and express it as a percentage of the theoretical amount of CO2 (TCO2) that could have been produced if complete biodegradation (mineralization) of the test substance occurred.

EXPERIMENTAL DESIGN
The test contained one control, one reference, and one treatment group. Each group contained triplicate test chambers. The control group was used to measure the background CO2 production of the inoculum and was not dosed with a carbon source. The reference group was dosed with canola oil, a substance known to be biodegradable, at a concentration of 10 mg carbon (C)/L. Treatment chambers were dosed with the test substance at a concentration of 10 mg C/L.

The test substance was administered to the treatment group test chambers by direct weight addition. The dosing amount was calculated based on measured carbon values. Direct weight addition is the most appropriate route of administration of substances that are relatively insoluble in water.

Reference Substance
Pure Wesson canola oil was used as a reference substance to check the activity of the inoculum. The canola oil used in this test was obtained from Food Lion #1289 (Baston, MD) and was assigned lot number 10-7-97. The reference substance was administered to the reference group test chambers by direct weight addition.

Test Medium
The test medium was a modified biochemical oxygen demand (BOD) test dilution water and was prepared using high quality water.

Test Apparatus and Conditions
The test chambers were 4-liter amber glass vessels. The air entering the chambers was passed through Drierite to remove ambient moisture and scrubbed using Ascarite to produce CO2 free air. The air exiting the test chambers was passed through a series of three gas washing bottles each containing approximately 100 mL of 0.5 N KOH to trap the CO2 that had evolved within the chamber. An. additional set of gas washing bottles that were not connected to a chamber were maintained concurrently with the traps connected to the chambers. The amount of CO2 detected in these traps was subtracted from the blank control traps to determine the amount of CO2 produced by the blank control. The test was conducted at 20±3°C. Magnetic
stirrers were employed to mix the contents of the chambers. The stirrers were cycled on and off approximately every 15 minutes to prevent heating of the stirrer motors. The chambers were identified by project number, test substance ID, test concentration and chamber number.

Preparation of Test Chambers:
1) 2470 ml of Nanopure water (ASTM type I)
2) 3 ml of ammonium sulfate solution (4.0%)
3 ml calcium chloride solution (2.75%)
12 ml of ferric chloride solution (0.025%)
3 ml of magnesium sulfate solution (2.25%)
30 ml of phosphate buffer ((pH 7.2)
3) 30 ml of the composite inoculum

All test chambers were aerated with CO2 free air for approximately 24 hours at a rate of 50-1 00 mL per minute to purge the system of CO2. After the aeration period, the flow of CO2 free air was stopped, and three CO2 traps each containing approximately 100 mL of 0.5 N KOH were connected to the exit air lines of each test chamber. A sufficient amount of the reference substance to achieve 10 mg CIL was added to the reference group chambers. A sufficient amount of test substance to achieve 10 mg CIL was added to the treatment group chambers. The final volume within all chambers was 3000 mL by the addition of Nanopure water.

Biodegradation Test Initiation
The biodegradation test was started by bubbling CO2 free air through the test media at a rate of 50-100 mL per minute. The CO2 produced from the degradation of organic carbon sources within each test chamber was trapped as K2C03 in the KOH solution and measured using a carbon analyzer.

Sample Collection and Analysis
The CO2 traps were removed for analysis on Days 4, 7, 12, 14, 19,22, and 29. The CO2 trap nearest the test chamber was removed and analyzed for inorganic carbon. The two remaining traps were placed one position closer to the test chamber and a new trap was placed on the end of the series.

Test Termination
On the 28th day of the test an aliquot of the contents of each test chamber was removed and the pH determined. The contents of each chamber were then acidified by the addition of 3 mL of concentrated hydrochloric acid to drive off inorganic carbonate. The chambers were aerated overnight and then the trapping solutions closest to the test chambers was analyzed for inorganic carbon.

Calculations:
The results of the carbon analyses were converted to mg CO2 using the following equation:

mg CO2 = result (mgC/L) X vol of KOH (L) X 3.67 mg CO/mg C

The cumulative mg of CO2 for the test and reference substances were corrected for the amount of CO2 evolved by control, using the following equation:

Cumulative mg CO2 evolved = the sum of mg CO2 test - mean sum of mg CO2 control

The percent of theoretical CO2 produced was calculated using the following equation:

%TCO2 = [mg CO2 produced/((mg carbon in test)/(3.67 mg CO2/mg carbon))] X 100

Reference substance:

other: Pure wesson canola oil

Test performance:

The temperature range recorded during the test was 19 to 21°C, within the range specified in the protocol. The result of the standard plate count performed on the inoculum prior to the. 14-day adaptation period was 1.79 E+05 CFU/mL. The result of the standard plate count performed on the composite inoculum
after the adaptation period was 1.98 E+04 CFU/mL. The pH of the test solutions at test termination are presented in Table 1. The results of the analysis of the CO2 trapping solutions are presented in Table 2 . The cumulative amounts of CO2 produced within each chamber are presented in Table 3. The percentages of
theoretical carbon dioxide production are presented in both tabular and graphical forms in Table 4 and Figure 4, respectively.

All tables and figures are attached in back ground material section.

The control chambers evolved an average of 3.6 milligrams of CO2 over the test period. This value has been corrected for the amount of CO2 in the trapping solution since potassium hydroxide solution, even when freshly prepared, contains carbonates. The amount of CO2 evolved by the control chambers did not exceed the 17 mg/L value considered the acceptable limit for CO2 evolution tests (I).

Parameter:

% degradation (CO2 evolution)

Value:

9.6

St. dev.:

3

Sampling time:

28 d

Details on results:

The test item attained an average percent biodegradation value of 9.6% (standard deviation 3.0) with an average final pH value of 6.37.

Results with reference substance:

The viability of the inoculum and validity of the test were supported by the reference substance, canola oil, degrading an average of approximately 77%.

Validity criteria fulfilled:

yes

Interpretation of results:

other: not readily biodegradable

Conclusions:

The test material attained and average of 9.6% degradation after 28 days (standard deviation 3.0) and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Executive summary:

Introduction and Method

This test method was designed to measure the degree of aerobic aquatic biodegradation of a lubricant or components of a lubricant by measuring evolved carbon dioxide (CO2). In the CO2 test, inoculated mineral medium is dosed with a known amount of test substance as the nominal sole source of organic carbon and aerated with CO2-free air. The CO2 produced from the mineralization of organic carbon within the test chambers is displaced by the flow of CO2 -free air and trapped as K2C03 in KOH trapping solution. The amount of CO2 produced by the test substance (corrected for that evolved by the blank inoculum) is expressed as a percentage of the theoretical amount of CO2 (TCO2) that could have been produced ,if complete biodegradation of the test substance occurred. Test materials that achieve a high degree of biodegradation in this test may be assumed to easily biodegrade in many aerobic aquatic environments. A low yield of CO2 does not necessarily mean that the test substance will not biodegrade under relevant environmental conditions but that additional testing may be needed to establish biodegradability

Results and Conclusion

The test material attained an average percent biodegradation of 9.6 (standard deviation 3.0). The average final PH was 6.37.

Description of key information

9.6% degradation was observed after 28 days, test substance was not considered to be readily biodegradable; study performed in line with OECD 301 C, ASTM Standard Test Method D 5864-95 and EPA OPPTS 796.3260; Haberlein and Schafer, (1998).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

A ready biodegradability test was designed to measure the degree of aerobic aquatic biodegradation of a lubricant or components of a lubricant by measuring evolved CO₂. In the CO₂ test, inoculated mineral medium was dosed with a known amount of test substance as the nominal sole source of organic carbon and aerated with CO₂-free air. The CO₂ produced from the mineralization of organic carbon within the test chambers is displaced by the flow of CO₂-free air and trapped as K₂CO₃ in KOH trapping solution. The amount of CO₂ produced by the test substance (corrected for that evolved by the blank inoculum) is expressed as a percentage of the theoretical amount of CO₂ that could have been produced, if complete biodegradation of the test substance occurred. Test materials that achieve a high degree of biodegradation in this test may be assumed to easily biodegrade in many aerobic aquatic environments. A low yield of CO₂ does not necessarily mean that the test substance will not biodegrade under relevant environmental conditions but that additional testing may be needed to establish biodegradability. Under the conditions of the test, the test substance attained an average biodegradation of 9.6% ± 3.0 and was not considered to be readily biodegradable.