Ethoprophos

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 1985-12-04 to 1986-07-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

1981

Deviations:

yes

Remarks:

See "Principles of method if other than guideline".

Principles of method if other than guideline:

The performance of the study requires 14C-test item. However, since no 14C-labelled test item was available, the test procedure had to be modified as follows:
- The test medium consisted of ditch water with a small amount of ditch sediment.
- The biodegradation was determined by monitoring three parameters, namely the oxygen uptake of the degrading micro-organisms, the concentration of test item in water (determined by chemical analysis), and the concentration of dissolved organic carbon (DOC) in the water.
- The sediment concentration had to be reduced to less than the specified 10 g/L, because of the oxygen consumption in the blank (medium and sediments without test item). A normal sediment concentration might also have interfered with the residue analysis, leading to higher estimates of primary biodegradation of the test item than actually occurred.

In view of the reduced initial biomass and higher relative concentration of test substance, the degradation potential of the modified test is expected to be lower than that of the method using medium with the normal amount of 10 g of dry sediment per litre.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethoprophos

EC Number:

236-152-1

EC Name:

Ethoprophos

Cas Number:

13194-48-4

Molecular formula:

C8H19O2PS2

IUPAC Name:

ethyl bis(propylsulfanyl)phosphinate

Test material form:

liquid

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

natural water / sediment: freshwater

Details on inoculum:

- Source of ditch water and sediments:
1. The ditch surrounding the premises of TNO-Zuidpolder at Delft, representing a non-polluted ditch (TNO inoculum).
2. A site in the 'Kromme Rijn' at Odijk, representing a polluted ditch (KR inoculum).
- Preparation of inoculum for exposure: The sediment was sieved to remove coarse particles before being added to the ditch water. Its dry weight was determined by drying subsamples at 105 °C for 3 hours.
- Pretreatment: analysed for pH and content of organics, calcium carbonate, silt, clay and sand
- Concentration of sludge: Samples of 5 or 10 litres of water were inoculated with 2 mL of sludge suspension per litre (amounting to 161 mg and 264 mg, respectively, for the two sludge types).
- Water filtered: yes
- Type and size of filter used: coarse paper filter

Duration of test (contact time):

82 d

Details on study design:

TEST CONDITIONS
- Composition of medium: ditch water and sediment
- Solubilising agent: no
- Test temperature: 20 °C

TEST SYSTEM
- Culturing apparatus: 300 mL BOD bottles
- Number of culture flasks/concentration: 4 replicates per treatment or toxicity control, control series with acetate in duplicate
The following additions were made to the different bottles:
- 8 mg of test item per litre
- 20 mg of test item per litre
- 40 mg of sodium acetate per litre, as a control on inoculum activity
- 40 mg of sodium acetate and 8 mg of test item per litre as a control of possible toxic effects
- 40 mg of sodium acetate and 20 mg of test item per litre, also a toxicity control
- Measuring equipment: OXI 191 oxygen electrode for O2 comsumption, Oceanographic International Corp. Model 524 TOC-analyser for DOC determination, chemical analysis by gas chromatography
- Test performed in closed vessels due to significant volatility of test substance: yes, stoppered with 10 to 15 mL air headspace

SAMPLING
- Sampling frequency: 10 mL
- Sample storage before analysis: 10 mL samples for DOC determination were stored in scintillation glass vials at -20 °C. The remaining contents of the bottles was acidified with dilute sulphuric acid (pH < 2) and cold-stored in the dark.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (DOC removal)

Value:

>= 65 - <= 100

Sampling time:

82 d

Remarks on result:

other: 20 mg test item/L in KR inoculum

% Degradation 2

Parameter:

% degradation (DOC removal)

Value:

>= 0 - <= 5

Sampling time:

82 d

Remarks on result:

other: 20 mg test item/L in TNO inoculum

% Degradation 3

Parameter:

% degradation (DOC removal)

Value:

>= 54 - <= 58

Sampling time:

82 d

Remarks on result:

other: 8 mg test item/L in TNO inoculum

% Degradation 4

Parameter:

% degradation (DOC removal)

Value:

>= 85 - <= 95

Sampling time:

82 d

Remarks on result:

other: 8 mg test item/L in KR inoculum

Any other information on results incl. tables

ACTIVITY AND TOXICITY OF INOCULUM
The biodegradability of acetate was used as a measure of the microbial activity of the inoculum and its possible inhibition by the presence of high concentrations of test substance. 

Table 1. Results of the control test, estimating the effect of the test item on the oxidation of sodium acetate.  The results are expressed as percentage oxidation of acetate after 8 and 14 days, respectively. The TOD of acetate is 0.59 mg O2/mg, and 40 mg of acetate was added per litre. The results are the mean value for four bottles, with the highest and lowest value in parentheses.

Test item mg/L (nominal)	% oxidation of acetate
	TNO		KR
	8 days	14 days	8 days	14 days
0	29.1 (22.7 - 38.2)	24.5 (11.0 - 36.8)	26.8 (20.3 - 29.1)	26.3 a) (11.7 - 36.8)
8	21.1 (17.9 - 26.5)	45.6 (24.4 - 65.9)	32.1 (26.2 - 38.5)	55.2 (41.7 - 65.5)
20	26.9 (20.2 - 32.7)	13.8 (10.5 - 16.2)	38.7 (31.4 - 50.0)	42.9 (34.0 - 55.2)

a) three valid values

 

The biodegradation of acetate itself had a slower progression than expected. This was due mainly to the erratic results after 14 days, indicating that some bottles may not have been fully air-thight during the test. There was a marked stimulation of the oxygen consumption in the presence of the test item, especially after 14 days. In some of these bottles the oxygen consumption, based on acetate degradation, reached levels indicating complete degradation within 14 days (> 60% oxidation).

The test item had no significant inhibiting effects on the degradation of acetate. However, the low oxidation with 20 mg/L of test substance and TNO inoculum cannot be explained properly. The DOC concentration as well as that of the test item were determined in the bottles of the control test.

Table 2. Reduction of DOC concentration in ditch water after 8 and 14 days incubation of the control test. The added amount of acetate (40 mg/L) corresponds to 11.5 mg DOC/L.

Experiment	DOC reduction (mg/L)
	TNO		KR
	8 days	14 days	8 days	14 days
Inoculum 40 mg Na-acetate 40 mg Na-acetate + 8 mg test item 40 mg Na-acetate + 20 mg test item	0.9 12.5   10.9 9.1	0.2 12.6   10.8 9.7	0 11.8   10.6 11.1	0.4 12.0   11.1 11.2
8 mg/L test item 20 mg/L test item	0 -	<0.1 -	0 0.3	- -

The results show that the two inocula cause full degradation of acetate within a week. The lower acetate degradation in the presence of 20 mg of test item per litre indicates an inhibition of the inoculum activity. The test item concentration did not decrease significantly during this part of the test.

 

BIODEGRADATION
Oxygen uptake
The determination of oxygen uptake was invalidated by the leakage of the test bottles. For this reason, oxygen uptake data only after 8, 14 and 82 days was reported. No initial biodegradation of the test item was found. After 82 days some oxidation of the test item could be found in tests with KR-inoculum. With 20 mg tets item/L, a maximum oxidation of 39 % of the TOD was found, indicating substancial mineralization of the test item.

DOC- and test item degradation
The biodegradability of the test item had to be asessed on the basis of DOC and test item concentrations as a function of time due to the lack of reliable oxygen consumption results. With a few exceptions only single series were analysed. Nevertheless, the comparison of the two types of measurements (assuming that the test item contains 0.40 mg DOC/mg) together with the duplicates in the control series showed a very satisfactory reproducibility of the determinations.
After 82 days the degradation reaches 11 - 14 % in the bottles inoculated with TNO sludge, and 31 to 93 % in the bottles inoculated with KR sludge. The degradation is clearly stimulated by the higher concentrations of test item with KR-inoculum. The TNO inoculum causes hardly any degradation. The KR-inoculum, however, causes the degradation rate to increase sharply between 57 and 82 days, leading to nearly complete primary degradation.

Table 3. Percentage primary degradation of the test item at two concentrations (8 and 20 mg/L) and with two inocula (TNO and KR) calculated on basis of the results of specific analysis.

Time (days)	TNO inoculum		KR inoculum
	8 mg/L	20 mg/L	8 mg/L	20 mg/L
8	0	-	0	5
14	3	-	-	-
21	8	-	0	5
43	12	-	-	-
56	8	-	0	20
82	14	11	31; 63	93; 82

Table 4. Percentage biodegradation of the test item at two concentrations (8 and 20 mg/L) and with two inocula (TNO and KR) determined as the reduction of DOC. The DOC concentrations in bottles with the test item were corrected for the DOC concentration in bottles without test item.

Time (days)	TNO inoculum		KR inoculum
	8 mg/L	20 mg/L	8 mg/L	20 mg/L
0	0	0	0	0
8	0	0	26	4
14	23	5	0	7
20	4	0	10	4
43	15	0	15	23
57	19	0	0	19
82	54; 58	5; 0	95; 85	65; 100; 90; 99

In the test with KR-inoculum the DOC degradation is similar to the primary test item degradation although somewhat higher after 82 days. This is also the case in the test of the high test item concentration with TNO-inoculum. In the test of the low concentration, however, the DOC degradation after 82 days is much higher than can be explained by the primary degradation of the test item.

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Interpretation of results:

not readily biodegradable

Conclusions:

The test item's biodegradation depends on the used inoculum. After a long lag phase a primarily mineralisation of the test item up to 100 % was observed after 82 days exposure period.

Executive summary:

The biodegradation of the test item was determined in ditch water containing about 200 mg of ditch sediment per litre according to OECD 301B. Two types of sediments were used, one from the TNO premises (TNO), representing a non-polluted ditch, and one from the 'Kromme Rijn' (KR), representing a polluted one.
In two experiments, 8 and 20 mg of the test item (nominal), respectively, were added to a 1 L of ditch water, and the biodegradation of the substance was monitored by determination of the oxygen consumption, the concentration of the test item, and that of dissolved organic carbon (DOC) during 82 days.
The results of the determination of oxygen consumption were less conclusive for the determination of the biodegradability, although it was clear that there was no biodegradation during the first 2 weeks, and that there might be a substantial mineralization of the test item in KR-inoculated water after 82 days. Furthermore, the oxygen consumption, together with the DOC determinations in the acetate controls, showed that there were few toxic effects, except possibly in TNO-inoculated water, and that the activity of the two inocula was comparable and adequate. The oxygen consumption indicated a marked stimulation of the degradation following the addition of the test item. Analysis of the test item, however, revealed that this stimulation was not due to its degradation.
In TNO-inoculated water, primary degradation of the tetst item was slight, about 13 percent in 82 days.
In KR-inoculated water, primary degradation was concentration-dependent, for the most part taking place between 57 and 82 days. With 8 mg/L test item, the primary degradation reached 31 to 63 %; and with 20 mg/L - 82 to 93 %. The primary degradation was followed by a similar DOC-reduction, even exceeding the primary degradation because the tets item apparently stimulates microbial activity generally. In conclusion, the test item was mineralized, without production of significant amounts of metabolites. The difference in degradative power between the two inocula may be due to the much higher content of organic matter of the TNO inoculum. Organic matter may prevent rapid degradation of the test item until it has itself been mineralized (diauxic effect).