Prometryn

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:

28 Oct 1997 to 17 Dec 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Paris 17/07/1992

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Activated sludge collected from the sewage treatment plant of CH-4153 Reinach on November 17, 1997. The pH after collection was 7.7. The preparation was carried out according to the method described in the guideline. The concentration of the inoculum in the test was 22.9 mg sludge/L.

Duration of test (contact time):

29 d

Initial conc.:

17.3 other: mg ThOC/L

Based on:

ThCO2

Remarks:

replicate 1

Initial conc.:

17.1 other: mg ThOC/L

Based on:

ThCO2

Remarks:

replicate 2

Initial conc.:

17.9 other: mg ThOC/L

Based on:

ThCO2

Remarks:

toxicity control

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

DESIGN AND PROCEDURE
- Vessels: 2 Liter flasks (dark brown glass) equipped with gas inlet and magnetic stirrer.
- Reconstituted Water: The reconstituted water was prepared according to the method described in the guideline.
- Temperature: 21 ± 2°C
- Aeration: around 25 mL/min purified from carbon dioxide,
- CO2-Scrubbers: approximately 250 mL scrubbers with gas inlet tubes equipped with sintered glass tips containing 200 mL 0.05 N sodium hydroxide.
- Only one CO2 scrubber was used per test bottle. Theoretically, a solution of 0.05 M NaOH is sufficient to trap at least two times more CO2 than the maximum ThCO2 which can evolve in each test bottle (including CO2 possibly evolved from the bacteria e.g., endogenous respiration). Moreover, experimentally it was confirmed that no measurable CO2 carry over has ever occurred with the scrubbers used.

SET UP OF FLASKS
- Flasks 1 and 2: test substance and inoculum
- Flasks 3 and 4: test medium and inoculum (inoculum blank)
- Flasks 5 and 6: reference substance and inoculum (procedure control)
- Flask 7: reference substance, test substance and inoculum (control of toxicity and inhibition of the bacteria's activity by the tests substance)

TEST CONCENTRATIONS
- Test substance : Replicate 1: 52.1 mg test substance/1.5L = 17.3 mg ThOC / L, Replicate 2: 51.6 mg test substance/1.5L = 17.1 mg ThOC / L
- Reference substance: 15 mg DOC/L
- Toxicity control: For the toxicity control, the reference amount was 15.0 mg DOC/L and the test substance amount was 17.9 mg ThOC/L.
- Preacclimation: Before application, the inoculum was pre acclimated to the test medium overnight. The aeration was performed inside 1.2 L water, containing the amount of mineral salts for 1.5L.
- Final volumes: The final volume was made up to 1.5 L with a 300 mL mixture made from the test substance (the above mentioned amount) in bidistilled water. This mixture was homogenized by ultrasonification for 1 minute and added to the test vessel, which was immediately connected to the CO2 scrubber.

DURATION AND SAMPLING
The test duration was 29 days. During this time die evolved CO2 trapped by the 0.05 N NaOH solution of the blank and test substance was measured at 0, 3, 6, 8, 10, 13, 16, 20, 24, 28 and 29 days. The last measurement was performed after acidification of the test medium with 1 mL of concentrated hydrochloric acid.

Reference substance:

benzoic acid, sodium salt

Test performance:

The cumulative amount of CO2 in the blank flasks were 30.8 and 32.6 mg/1.5L, equivalent to 8.4 and 8.9 mg TOC/1.5 L. The total CO2 evolution in the inoculum blank at the end of the test did not exceed 60 mg/1.5 L. These results show that the test system was working properly and no CO2 leaks were present. The biodegradation of the reference substance reached the level for ready biodegradability in a 10-day window. After 10 days of incubation 75 % of the reference compound, sodium benzoate, had degraded, therefore the test is valid.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

29 d

Details on results:

The percent biodegradation, based on CO2 evolution versus time for the substance and the reference substance is presented in Tables 1 to 2 in “Any other information on results incl tables”. The theoretical amount of CO2 in the test flasks with the test substance alone were 95.0 and 94.1 mg/1.5L, equivalent to 25.92 and 25.68 mg TOC/1.5 L.

VALUES
The high rate of CO2 evolution in the toxicity control (reference + test substance), which was higher than the CO2 evolution in the reference test flask, indicates that the test substance did not inhibit the biodegradation of the reference substance. The biodegradation of the test substance at the end of the test was -0.5 (8.2-8.7* ) and - 1.5 (7.3-8.7* ) mg TOC/1.5L (see Tables 1 to 2 in “Any other information on results incl tables”), respectively. Therefore the biodegradation of the test substance was: 0 % in 29 days. (*: biodegradation of the test substance minus blank control)

Table 1. CO2 evolution and biodegradation of the controls: together Blank, reference substance and reference+test substance.

CO2 evolution measured as mg C							Biodegradation
day	Blank			Reference
	1	2	mean	1	2	mean	Reference+Test substance	Reference	Reference+Test substance
	[mg]	[mg]	[mg]	[mg]	[mg]	[mg]	[mg]	[%]	[%]*
0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0	0
3	2.7	2.6	2.7	13.7	13.7	13.7	15.3	49	56
6	4.7	5.0	4.9	19.9	19.3	19.6	17.8	65	57
8	5.5	5.7	5.6	21.1	21.7	21.4	19.3	70	61
10	6.0	6.0	6.0	22.6	23.3	23.0	20.3	75	63
13	6.7	6.6	6.7	23.7	24.5	24.1	21.8	78	67
16	7.5	7.7	7.6	25.4	26.2	25.8	23.4	81	70
20	7.6	7.9	7.7	25.8	26.6	26.2	23.5	82	70
24	8.0	8.0	8.0	26.4	27.1	26.8	24.4	83	73
28	8.2	8.2	8.2	27.2	28.0	27.6	25.1	86	75
29	8.4	8.9	8.7	27.0	28.1	27.6	25.6	84	75

* The biodegradation of the "reference + test substance" control is calculated with the applied

amount of the reference alone, i.e., 15 mg DOC/L

 

Table 2. CO2 evolution and biodegradation of the test substance

day	CO2 evolution measured as mg C		Biodegradation % of the test substance
	1	2	1	2	mean
0	0.0	0.0	0	0	0
3	2.8	2.6	1	0	0
6	4.7	4.2	-1	-2	-2
8	5.2	4.8	-1	-3	-2
10	5.7	5.2	-1	-3	-2
13	6.4	5.7	-1	-4	-2
16	7.6	5.7	0	-7	-4
20	7.2	6.4	-2	-5	-4
24	7.6	6.5	-2	-6	-4
28	8.0	6.6	-1	-6	-3
29	8.2	7.3	-2	-5	-4

Remark: Negative degradation values, when obtained in a test, are a consequence of the comparison of values obtained in the blank (endogenous respiration leading to a carbon dioxide evolution) and the low values obtained in the presence of the test substance.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The mineralisation of the substance under these test conditions was 0 % in 29 days. Based on the results of this test alone and using the classification range given in Annex VI of directive 67/548/EEC, the ecotoxicological classification is: “not readily biodegradable”.

Executive summary:

This study investigated the ready biodegradability of the substance in the CO2-Evolution Test in activated sludge collected from a sewage treatment plant according to the OECD TG 301B and in compliance with GLP over a period of 29 days. A measured volume (1.5 L) of inoculated mineral medium containing 52.1 mg resp. 51.6 mg test substance (17.3 resp.17.1 mg TOC/L) as the nominal sole source of organic carbon was aerated by the passage of carbon dioxide-free air at a controlled rate in diffuse light. Degradation was followed over 29 days by determining the carbon dioxide produced, which was trapped in sodium hydroxide and measured as inorganic carbon by an analyser. The amount of carbon dioxide produced from the test chemical (corrected for that derived from the blank inoculum) was expressed as a percentage of theoretical carbon dioxide. A reference (sodium benzoate) and a toxicity control (sodium benzoate plus test substance) were used as controls. The inoculum concentration in the test bottles was 22.9 mg suspended solids/L. The pH of the sludge after collection was 7.7. In the test with the substance the temperature was maintained constant at 21 ± 2 °C. The biodegradability of the reference substance after 10 days was 75%, demonstrating the suitability of the test system. The biodegradation of the test substance calculated as percentage of measured amount of inorganic carbon over the theory was 0% in 29 days. Based on the results, the test substance was considered to be “not readily biodegradable”. 

Description of key information

The substance is not biodegradable under the test conditions, OECD TG 301B, Grade 1998.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

This study investigated the ready biodegradability of the substance in the CO2-Evolution Test in activated sludge collected from a sewage treatment plant according to the OECD TG 301B and in compliance with GLP over a period of 29 days. A measured volume (1.5 L) of inoculated mineral medium containing 52.1 mg resp. 51.6 mg test substance (17.3 resp. 17.1 mg TOC/L) as the nominal sole source of organic carbon was aerated by the passage of carbon dioxide-free air at a controlled rate in diffuse light. Degradation was followed over 29 days by determining the carbon dioxide produced, which was trapped in sodium hydroxide and measured as inorganic carbon by an analyser. The amount of carbon dioxide produced from the test chemical (corrected for that derived from the blank inoculum) was expressed as a percentage of theoretical carbon dioxide. A reference (sodium benzoate) and a toxicity control (sodium benzoate plus test substance) were used as controls. The inoculum concentration in the test bottles was 22.9 mg suspended solids/L. The pH of the sludge after collection was 7.7. In the test with the substance the temperature was maintained constant at 21 ± 2 °C. The biodegradability of the reference substance after 10 days was 75%, demonstrating the suitability of the test system. The biodegradation of the test substance calculated as percentage of measured amount of inorganic carbon over the theory was 0% in 29 days. Based on the results, the test substance was considered to be “not readily biodegradable”.