Chlorpyrifos

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch Number: YH2-132673-071-te
Radiochemical Purity: >96.8%
Specific Activity: 30.3 mCi/mmol, corresponding to 3.20 MBq/mg

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

- Details on collection: The test water was collected from a typical natural pond (Möhlin, AG, Switzerland) system not receiving human or agricultural effluent discharges, being located away from obvious human activity. The water was sampled at a depth of about 30 cm and approximately 1 m from the embankments of the pond.
- Storage conditions: 5°C
- Storage length: 18 days
- Temperature at time of collection: Main study: 12.5°C; follow-up test: 1.9°C
- pH at time of collection: Main study: 7.89; Main study: 7.89
- Redox potential (mv) initial: Main study: 189; Main study: 168
- Oxygen concentration (mg/l) initial: Main study: 7.62; Main study: 4.8
- Hardness (°dH): Main study: 14.2; Main study: 15.1
- Dissolved organic carbon (mg C/L): Main study: 13.6; Main study: 13.7
- BOD (mg/L): Main study: <0.4; Main study: <0.5
- TOC (mg C/L): Main study: 13.6; Main study: 13.7
- Biomass (e.g. in mg microbial C/100 mg, CFU or other):
- Water filtered: Yes
- Type and size of filter used: 0.2 mm sieve

Duration of test (contact time):

61 d

Initial conc.:

12.1 µg/L

Based on:

test mat.

Initial conc.:

126 µg/L

Based on:

test mat.

Initial conc.:

130 µg/L

Based on:

test mat.

Initial conc.:

102 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

TEST CONDITIONS
- Test temperature: 20 ± 2°C

TEST SYSTEM
- Culturing apparatus: All-glass metabolism flasks (inner diameter: about 5.3 cm, volume:approx. 500 mL)
- Number of culture flasks/concentration: 16 (main test); 4 (follow-up test)
- Number of culture flasks for sterile control: 10 (main test); 4 (follow-up test)
- Method used to create aerobic conditions: Each flask was equipped with an air inlet and outlet

- Test performed in closed vessels due to significant volatility of test substance: Yes
- Details of trap for CO2 and volatile organics if used: 50-60 mL 2M NaOH solution

SAMPLING
- Sampling frequency: 0, 1, 5, 14, 41, 61 days for test substance and 10, 20, 40 for sterile control (main test) ; 20, 40 (follow-up test)

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Abiotic sterile control: Ten (main test) and four (follow-up test) sterile control test flasks were dosed at approximately 100 μg/L [14C]test substance

Reference substance:

other: benzoic acid

Key result

Compartment:

natural water

DT50:

46 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20 °C

Remarks on result:

other: 12.1 µg/L

Key result

Compartment:

natural sediment

DT50:

21 d

Type:

(pseudo-)first order (= half-life)

Temp.:

20 °C

Remarks on result:

other: 126 µg/L

Transformation products:

yes

No.:

#1

Evaporation of parent compound:

yes

Volatile metabolites:

yes

Remarks:

CO2 and volatile organic carbon

Details on results:

MAJOR TRANSFORMATION PRODUCTS
Main test: One major metabolite M1 was observed in the study, which was assigned by HPLC and TLC co-chromatography to the reference compound R4 (3,5,6-trichloro-2-pyridinol). In the FTL samples, mean amounts of M1 increased from 2.0% (Day 0) to 45.1% AR (Day 61). In the FTH samples, M1 increased from 0.5% to 59.7% AR (Day 61). In the sterile samples, 22.6% AR of M1 were found on Day 40, corresponding Day 41 values in the FTL and FTH samples were 20.8% and 34.1% AR, respectively.
Follow-up test: M1 was also formed in both the FTH-2 and the FS-2 systems with up to 11.1% AR

MINOR TRANSFORMATION PRODUCTS
Up to six smaller metabolite signals were found by HPLC, only M2 (R5, Chlorpyrifos oxon) showed an abundance of up to 3.2% between sacrifice days 0 and 5. All other metabolites can be considered as minor (<1% in all systems). One of them co-eluted in HPLC with the reference standard R6 (Desethyl Chlorpyrifos).

MATERIAL BALANCE:
Main test: Mean mass balances and standard deviations were 87.7 ± 6.3%, 91.1 ± 11.8%, and 66.1 ± 11.4%, respectively. In all three test groups, the mean mass balance decreased towards the end of the incubation period to 81.1% (FTL, Day 61), 71.5% (FTH, Day 61), and 51.7% (FS, Day 40) suggesting a progressive loss of material e.g. by evaporation.
In the follow-up test, which was performed to investigate the reasons for the low mass balance in the main test, the mean recoveries were 103.5 ± 2.3% and 102.5 ± 4.7% for the non-sterile and sterile samples, respectively.

Results with reference substance:

The reduction of [14C]CO2 formation in the solvent–treated flasks is considered as an artifact. The [14C]Benzoic acid samples in this study were treated four months after sampling and application of the main study, which can explain this increased sensitivity to the co-solvent. For comparison, data was shown from the [14C]benzoic acid control experiment performed in a pretest this study, acetonitrile had no effect on mineralization. [14C]CO2 formation in this batch of water, as measures on Day 17, was 71.5% in the blanks and 84.6% AR in the solvent treated flasks.

Validity criteria fulfilled:

yes

Conclusions:

Based on the kinetics results, test substance has a faster dissipation rate in the surface water system dosed at the higher concentration.

Executive summary:

The time course and concentration dependency of the biotransformation of [14C]test substance in aerobic surface water (“pelagic test”) was investigated at 22.0 ± 0.2 °C in the dark for up to 61 days. For the main test, the test concentrations were 12.1 μg/L (low concentration viable, FTL), 126 μg/L (high concentration viable, FTH), and 130 μg/L for the sterile controls (high concentration, sterile FS). After the main test, a follow up test was performed with additional non sterile (high concentration, viable, FTH-2) and sterile (high concentration sterile, FS-2) samples treated at an actual concentration of 102 μg/L. The purpose of the follow-up test was the investigation of the amount and nature of the volatile radioactivity which was observed in the main test.

All [14C]test substance test concentrations in the study were below the water solubility limit of test substance (1050 μg/L).

To confirm the microbial activity of the test water, reference and solvent control samples (FC and SC, respectively) were treated with [14C]Benzoic acid at concentrations of 9.8 μg/L. The experiment was conducted in accordance with the OECD Guideline 309.

Both main and follow-up studies were performed using a negative pressure flow through test system. Glass metabolism flasks equipped with air inlets and outlets were in turn attached to a series of absorption traps for the sequestration of volatile radioactive species. ORBO™53 activated silica gel solid state tubes, followed by ethylene glycol liquid traps (~50 mL) were set up as the trapping systems for volatile organic components. The series of volatile traps were completed with 2 M aqueous sodium hydroxide (~ 60 mL) for the collection of any evolved [14C]carbon dioxide. During the incubation period, the metabolism flasks were continuously agitated via magnetic stirring. The test flasks were insulated from the magnetic stirrers using cardboard, in order to prevent mechanical temperature elevation.

The follow-up test was performed in the same set-up as the main test, with the addition of a polyurethane foam plug, which was placed into the outlet in the neck of the metabolism flask to allow a more efficient capture of organic volatiles.

Duplicate samples of each test system containing [14C]test substance were sacrificed and analyzed at the following sampling intervals: FTL and FTH test systems: 0, 1, 5, 14, 41, 61 days; FS test system: 10, 20, 40 days; FTL-2 and FS-2 test systems: 20 and 40 days.

During the incubation period, the test systems were continuously agitated through aeration and magnetic stirring.

Duplicate test flasks containing the reference substance [14C]Benzoic acid (FC) were sacrificed after 0, 3 and 14 days of incubation. Significant evolution of 14CO2 after 14 days, more than 50% of applied in the blank and 28% in the solvent control flasks confirmed the microbial activity of the test water. The reduction of the biological activity by acetonitrile is considered to be an artefact, as this effect was not observed in the pre-test to this study and in other batches sampled from the same source.

Mean mass balance results (± s.d.) as percentages of applied radioactivity [AR] for the main test samples FTL, FTH and FS test systems were 87.7 ± 6.3%, 91.1 ± 11.8%, and 66.1 ± 11.4%, respectively. In all three test groups, the mean mass balance decreased towards the end of the incubation period to 81.1% (FTL, Day 61), 71.5% (FTH, Day 61), and 51.7% (FS, Day 40) suggesting a progressive loss of material e.g. by evaporation.

For the FTL, FTH and FS test flasks, data generated from the elution/extraction of the ORBO™53 tubes indicated that insignificant quantities of radioactivity were associated with the solid state phase over the study exposure period.

Mineralization of [14C]test substance was negligible in the time frame of the study. Formation/evolution of other volatile components was insignificant with mean values equating to ≤ 0.9% AR at all sampling intervals for all FS test systems.

In the follow-up test, which was performed to investigate the reasons for the low mass balance in the main test, the mean recoveries were 103.5 ± 2.3% and 102.5 ± 4.7% for the non-sterile (FTH-2) and sterile samples (FS-2), respectively. Up to 95.7% (FTH-2, Day 40) of the radioactivity was found in the foam plugs and shown to be 100% unchanged [14C]test substance. These results show, that the low mass balance observed from the later time points in the main test is due to evaporation of unchanged parent compound. Therefore, the loss of test substance in the main test has no significant impact on the study results and conclusion. The surface water mineralisation results observed in the main test is valid and truly representative of the behavior of test substance in natural water system.

The amounts of unchanged [14C]test substance decreased throughout the incubation periods from 94.3% AR (0 h; mean) to 34.2% AR (Day 61; mean), 98.8 % AR (0 h; mean) to 9.4% AR (Day 61; mean) for the FTL and FTH test systems, respectively. In the FS system, 26.4 % unchanged parent were observed after 40 days.

One major transformation product, M1, was observed in all test systems, and identified through radio-HPLC co-chromatography and confirmatory radio-TLC chromatography as the analytical reference standard 3,5,6-trichloro-2-pyridinol. The amounts of minor metabolites remained under 1% of applied. Only Chlorpyrifos oxon M2 reached maximum concentrations of 2% and 3.2% AR on Day 1 in the FTL and FTH samples, respectively, but mean concentrations of M2 were below 1% on Day 5.

SFO best-fit for the test substance: DT50: 46 days (12.1 µg/L); DT50: 21 days (126 µg/L)

Description of key information

Study Type	Study Details	Value	Guideline	Reliability
Biodegradation in water: simulation testing on ultimate degradation in surface water	Simulation biodegradation test	DT50 (Low concentrations) = 46 days  DT50 (High concentrations) = 21 days	OECD 309 EPA OPPTS 835.4300	1