Paraquat-dichloride

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:

21 Jun 2018 to 27 Jun 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test substance was directly pipetted into each test flask which was filled with pure water

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: Municipal sewage treaetment plant
- Preparation of inoculum for exposure: the activated sludge was used as collected, but coarse particles were removed by settling for a short period (15 minutes) and then the upper layer decanted. During holding prior to use for one day (dose-response study) or two days (limit study), the sludge was fed daily with 50 mL synthetic sewage feed per litre and kept aerated at room temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

pH:

6.7 - 7.6 (measured at start and end of incubation period)

Dissolved oxygen:

6.1 - 8.8 mg O2/L

Nominal and measured concentrations:

- Nominal concentrations: 0 (control) 10, 32, 100, 320 and 1000 mg technical test material/L

Details on test conditions:

TEST SYSTEM
For both tests, an aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentration equivalent to 3 g/L on dry weight basis. This level gave a concentration of 1.5 g/L suspended solids in the test medium. In the dose-response study, where additionally the heterotrophic respiration was measured, the amount of water was reduced by the amount of the nitrification inhibitor ATU.

- Test vessel: Glass flasks of approximately 1 litre volume and Karlsruher flasks
- Fill volume: 250 mL
- Aeration: Yes, with compressed air (1.017 litre per minute)
- No. of vessels per concentration: 5
- No. of vessels per control: 6
- No. of vessels per positive control: 5
- Sludge concentration (weight of dry solids per volume): 1.5g/L
- Nutrients provided for bacteria: Synthetic sewage feed was prepared with 80 g peptone, 55 g meat extract, 15 g urea, 3.5 g NaCl, 2.0 g CaCl2.2H2O, 1.0 g MgSO4.7H2O and 14 g K2HPO4, filled up to 5 litre with deionised water
- Nitrification inhibitor used: Yes, N-allylthiourea (11.6 mg ATU/L)

PREPARATIONS OF THE TEST FLASKS
For each replicate of the total respiration rate (pre-test and main test (dose-response study)) a test solution with a final volume of 500 mL was tested per treatment in a glass flask. 16 mL synthetic sewage feed and an adequate volume of the test item or an adequate volume of the stock solution of the reference item were filled up with pure water to 250 mL before the start of the test. At the start of the test 250 mL activated sludge inoculum with a sludge concentration of 3.0 g/L suspended solids was added, first to two controls, then to the test solutions of the reference item, to further controls, to the test item and finally to additional controls. During 3 hour aeration period the flasks were stirred on a magnetic stirrer to maintain sludge flocs in suspension.

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: Deionised water with synthetic sewage feed

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Lighting: Daylight

WATER QUALITY MEASUREMENTS
The oxygen concentrations were determined at the start and at the end of the incubation period in at least one replicate of all test concentrations and controls. The pH value was determined at the start and at the end in at least one replicate of the test concentrations and controls. The water temperature was measured in one control medium at the start and the end of the incubation period.

EFFECT PARAMETERS MEASURED: oxygen consumption
For the measurement of the respiration rate a well-mixed sample of test medium from each flask was poured into a Karlsruher flask after exactly 3 hours incubation time and was not further aerated during measurement. The oxygen concentration was then measured with an oxygen electrode and recorded for about ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using fewer concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

RANGE FINDING STUDY
- Test concentrations: Nominal 10, 100 and 1000 mg test item/L (1 replicate for 10 and 100 mg test item/L and 3 replicates for 1000 mg test item/L). Six control replicates were used.
- Validation criteria met: Yes
- Results: In comparison to the inoculum controls the respiration rates of the activated sludge was inhibited by 24.1% and 32.3% at test item concentrations of 10 mg/L and 100 mg/L, respectively. The tested concentration of 1000 mg/L resulted in an inhibitory effect on activated sludge bacteria of 56.9% (mean). Concentrations exceeding 1000 mg/L nominal were not tested. Therefore no NOEC could be determined and a dose-response study was performed. Although the reference item validity criterion was not fulfilled, the interpretation of the results and the performance of a dose response study remain correct.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

29.62 mg/L

Nominal / measured:

nominal

Conc. based on:

other: pure test substance

Basis for effect:

inhibition of total respiration

Remarks on result:

other: recalculated value, expressed as pure substance, see ‘any other information on results incl. tables’ for respective calculation

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

65.09 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: Originial value as presented in the study report

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

4.55 mg/L

Nominal / measured:

nominal

Conc. based on:

other: pure test substance

Basis for effect:

other: total, heterotrophic and nitrification respiration

Remarks on result:

other: The NOEC could not be determined

Remarks:

recalculated value, expressed as pure substance, see ‘any other information on results incl. tables’ for respective calculation

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: total, heterotrophic and nitrification respiration

Remarks on result:

other: The NOEC could not be determined

Remarks:

Originial value as presented in the study report

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

72.99 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

other: Originial value as presented in the study report

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

319.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks on result:

other: Originial value as presented in the study report

Details on results:

- Total respiration: In comparison to the inoculum controls the respiration rates of the activated sludge were consistently inhibited up to test concentrations of 320 mg/L; the inhibition was 40.2%, 37.7%, 34.9% and 46.2% for test item concentrations of 10, 32, 100, 320 mg/L, respectively. A test item concentration of 1000 mg/L caused an inhibitory effect of 61.8%. Concentrations exceeding 1000 mg/L nominal were not tested.
- Heterotrophic Respiration (Respiration Rate and Inhibition): The inhibition of the heterotrophic respiration of the activated sludge showed a weak dose-response inhibition up to concentrations of 1000 mg/L; the inhibition was 30.9%, 44.0%, 48.0%, 52.4% and 56.9% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively.
- Nitrification Respiration (Respiration Rate and Inhibition): In comparison to the inoculum controls, the respiration rates of the activated sludge based on nitrification showed no clear dose-response inhibition; the inhibition was 56.7%, 26.4%, 11.5%, 35.1% and 70.5% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively.

Results with reference substance (positive control):

- Total respiration: In comparison to the controls the total respiration rate of the activated sludge was inhibited by 43.4% at the lowest nominal concentration of 1 mg/L. At the nominal concentrations of 4 and 16 mg reference item/L, the respiration rate was inhibited by 48.7% and 84.8%, respectively. The 3-h EC50 was determined to be 2.2 mg/L based on total respiration.
- Heterotrophic respiration: In comparison to the controls the heterotrophic respiration rate of the activated sludge was inhibited by 23.7% at the lowest nominal concentration of 1 mg/L. At the nominal concentrations of 4 and 16 mg reference item/L, the respiration rate was inhibited by 24.5% and 71.8%, respectively. The 3-h EC50 was determined to be 8.4 mg/L based on heterotrophic respiration.
- Nitrification respiration: In comparison to the controls the nitrification respiration rate of the activated sludge was inhibited by 78.4 % at the lowest nominal concentration of 1 mg/L. At the nominal concentrations of 4 and 16 mg reference item/L, the respiration rate was inhibited by 91.6% and 107.9%, respectively. The 3-h EC50 was determined to be 0.3 mg/L based on nitrification respiration.

Reported statistics and error estimates:

The EC50 including the 95 %-confidence limits of the reference item and test item was calculated by a 3-Parametric normal CDF (cumulative distribution function), non-linear regression without weighting; Optimization method: Levenberg-Marquardt.
For the determination of the NOEC of the test item, the Williams Multiple Sequential t-test (α= 0.05, one-sided smaller) was used after testing the normal distribution by the Shapiro Wilk’s test (α= 0.01) and the variance homogeneity by the Levene’s Test (α= 0.01).

Table: Influence of the test substance on total oxygen consumption of activated sludge in a 3-hour respiration inhibition test

Treatment	Conc. [mg/L]	Resp. Rate (1) [mg O2/(L*h)]	SD (1) %	Resp. Rate (1) [mg O2/g/ h]	SD (1) %	Inhibition (1) %	pH-values (2)		O2 conc. [mg O2/L] (2)
							start*	end*	start*	end*
control	---	33.9	3.1	22.6	2.1	---	6.8	7.4	6.4	7.4
3,5-DCP	1	19.2	2.5	12.8	1.7	43.4	7.0	7.7	6.2	8.5
3,5-DCP	4	17.4	1.8	11.6	1.2	48.7	7.0	7.5	6.3	8.3
3,5-DCP	16	5.2	0.5	3.4	0.4	84.8	6.8	7.1	6.5	8.8
Test item	10	20.3	1.8	13.5	1.2	40.2	6.8	7.3	6.5	8.2
Test item	32	21.1	0.9	14.1	0.6	37.7	6.8	7.3	6.2	7.8
Test item	100	22.1	2.8	14.7	1.9	34.9	6.7	7.3	6.8	7.8
Test item	320	18.2	0.7	12.2	0.5	46.2	6.8	7.6	6.6	8.4
Test item	1000	13.0	0.9	8.6	0.6	61.8	6.8	7.6	6.5	8.6

Coefficient of Variation of control: 9.2%  

(1) mean value of 5 replicates, 6 replicates in case of control

(2) mean value of 3 replicates for control; measured in replicate 1 for 3,5-DCP and test item

*) Start and end of 3-hour aeration

DCP: 3,5-Dichlorophenol

SD: standard deviation; Conc.: Concentration; Treat: Treatment; Resp.: Respiration

Table: Influence of the test substance on heterotroph oxygen consumption of activated sludge in a 3-hour respiration inhibition test

Treatment	Conc. [mg/L]	Resp. Rate (1) [mg O2/(L*h)]	SD (1) %	Resp. Rate (1) [mg O2/g/ h]	SD (1) %	Inhibition (1) %	pH-values (2)		O2 conc. [mg O2/L] (2)
							start*	end*	start*	end*
control	---	21.7	2.1	14.5	1.4	---	6.8	7.6	6.5	8.2
3,5-DCP	1	16.6	1.0	11.0	0.7	23.7	6.9	7.7	6.5	8.5
3,5-DCP	4	16.4	1.7	10.9	1.1	24.5	7.0	7.5	6.1	8.2
3,5-DCP	16	6.1	1.3	4.1	0.9	71.8	6.8	7.1	6.0	8.9
Test substance	10	15.0	1.7	10.0	1.1	30.9	6.8	7.7	6.7	8.2
Test substance	32	12.1	0.9	8.1	0.6	44.0	6.8	7.7	6.1	8.2
Test substance	100	11.3	0.7	7.5	0.4	48.0	6.7	7.6	6.6	8.4
Test substance	320	10.3	0.5	6.9	0.3	52.4	6.8	7.5	6.9	8.6
Test substance	1000	9.4	0.3	6.2	0.2	56.9	6.8	7.7	6.5	8.8

(1) mean value of 5 replicates, 6 replicates in case of control

(2) mean value of 3 replicates for control; measured in replicate 1 for 3,5-DCP and test item

*) Start and end of 3-hour aeration

DCP: 3,5-Dichlorophenol

SD: standard deviation ; Conc.: Concentration; Treat: Treatment; Resp.: Respiration

Calculation of key results

The concentrations of the test substance were expressed in technical test material, which relates to an aqueous solution of the registered substance (purity = 45.5%). The effect values are calculated by correction for the amount of water:

- EC50 (on inhibition of total respiration): 0.455 x 65.09 mg technical test material/L = 29.62 mg pure test substance/L.

- LOEC: 0.455 x 10 mg technical test material = 4.55 mg pure test substance/L.

Validity criteria fulfilled:

yes

Conclusions:

The 3-h EC50 was 65.09 mg technical test material/L for total respiration, 72.99 mg technical test material/L for heterotrophic respiration and 319.02 mg technical test material/L for nitrification respiration. As the confidence limits are wide, these values should be handled with care. The 3-h EC50 of 65.09 mg technical test material/L for total respiration value corresponds to 29.62 mg pure substance/L. The NOEC could not be determined.

Executive summary:

The influence of the test substance on the activity of activated sludge according to OECD TG 209 and in compliance with GLP criteria was investigated at concentrations of 3.2 mg, 10 mg, 32 mg, 100 mg, 320 mg and 1000 mg/L (dose-response study). The test substance had constantly toxic effects on total respiration activity of activated sludge after the incubation period of 3 hours for test item concentrations up to 320 mg/L. A test item concentration of 1000 mg/L caused increased inhibiting effects on total respiration. The heterotrophic respiration activity showed a weak dose-response inhibition up to concentrations of 1000 mg/L. The respiration rates of the activated sludge based on nitrification showed no clear dose-response inhibition. The 3-h EC50 values are determined to be 65.09 mg/L for total respiration, 72.99 mg/L for heterotrophic respiration and 319.02 mg/L for nitrification respiration. The NOEC could not be determined. The 3-h EC50 of 65.09 mg technical test material/L for total respiration value corresponds to 29.62 mg pure substance/L.

Description of key information

The 3-h EC50 and EC50 value is determined to be 29.62 mg pure substance/L (recalculated) in domestic activated sludge based on total respiration inhibition. The NOEC could not be determined.

Key value for chemical safety assessment

EC50 for microorganisms:

29.62 mg/L

Additional information

Table: Overview of available data on the toxicity to microorganisms:

Species	Guideline / GLP	Endpoint	Effect value	Comment	Reference
Activated sludge (domestic)	OECD TG 209 / GLP	3-h EC50	29.62 mg pure substance/L (recalculated)	Effect value based on total respiration inhibition. NOEC could not be determined.	Hammesfahr, 2018