Captan

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:

13 March to 24 July 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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical state and appearance: White powder
Supplier: ADAMA Makhteshim Ltd.
Lot/batch number: 941381319
Purity/strength: 96.5%
Date of analysis/certificate dated: 19 May 2015
Expiry date: 01 January 2020
Sample storage: Room temperature

Details on sampling:

- Concentrations:
Preliminary Range-finder test
The preliminary range-finder test included the following vessels, all containing activated sludge at a sludge solids concentration of 1500 mg/L and an excess of synthetic sewage feed:
Duplicate blank controls and single replicates of reference substance, 3,5-DCP, at 1.0, 2.0, 5.0, 10 and 25 mg/L.
Duplicate blank controls, single replicates of Captan Technical at 10 and 100 mg/L and triplicate Captan Technical at 1000 mg/L.
Nitrification inhibition vessels: all vessels contained 11.6 mg/L N-allylthiourea (ATU) to inhibit oxidation of ammonia to nitrite. Duplicate blank controls, single replicates of Captan Technical at 10 and 100 mg/L and triplicate Captan Technical at 1000 mg/L.

Definitive test
The definitive test included the following vessels, all containing activated sludge at a sludge solids concentration of 1500 mg/L and an excess of synthetic sewage feed:
Nominal 0.25, 0.64, 1.6, 4.0 and 10 mg/L concentrations of Captan Technical were prepared in replicates of five. For the reference substance (3,5-DCP), single replicates at 1.0, 2.0, 5.0, 10 and 25 mg/L and a blank control single replicate were prepared.
The first batch consisted of a set of reference substance flasks and three Captan Technical concentrations (0.25, 0.64 and 1.6 mg/L) with a control flask run with each set.
The second batch consisted of a set of reference substance flasks and two Captan Technical concentrations (4.0 and 10 mg/L) with a control flask run with each set.

- Sampling method:
For the definitive total respiration test (first and second batch) the respiration rate was measured in the control flask and four of the test or reference substance flasks, then a fresh sub-sample from the control flask had the respiration rate measured with the fifth test or reference substance flask.
On the second day the third batch consisted of a set of reference substance flasks with a control flask and nitrification inhibition vessels: all vessels contained 11.6 mg/L ATU to inhibit oxidation of ammonia to nitrite this consisted of five Captan Technical concentrations (0.25, 0.64, 1.6, 4.0 and 10 mg/L) with a control flask.

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge was obtained from Totnes Sewage Treatment Works, Totnes, Devon, UK on 13 March 2018 for the preliminary test, 27 March 2018 for the definitive total respiration test and 29 March 2018 for the definitive heterotrophic respiration test. This works treats sewage of predominantly domestic origin. At the laboratory, the activated sludge was settled and the supernatant decanted. The settled sludge was fed with 50 mL of OECD synthetic sewage feed per litre of sludge per day and aerated at room temperature, until it was used in the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 ± 2°C

pH:

7.5 ± 0.5

Nominal and measured concentrations:

Captan technical: 0.25, 0.64, 1.6, 4.0 and 10 mg/L (five replicates each)
Reference item: 1.0, 2.0, 5.0, 10 and 25 mg/L (single replicates)
Blank control (single replicate)

Details on test conditions:

PREPARATION OF STOCK SOLUTIONS
Due to the low water solubility of Captan Technical for the preliminary test a known weight was added to each flask by direct weigh method.
For the definitive tests a stock solution of Captan Technical was prepared at a nominal concentration of 3 mg/L in reverse osmosis (RO) water; this stock was observed to be a clear colourless solution. A known volume of this stock solution was used to dose the 0.25, 0.64 and 1.6 mg/L test flasks. Due to the solubility limit of Captan Technical the 4 and 10 mg/L test flasks had a known weight of Captan Technical added to each flask by direct weigh method.
REFERENCE SUBSTANCE
A nominal 100 mg/L stock solution of the reference substance, 3,5-DCP, was prepared in RO water.
TEST APPARATUS
The respiration rate of the test solutions was measured using the Strathkelvin Strathtox respirometer. This is an instrument for the measurement of the oxygen uptake rate of six samples simultaneously. It consists of a six place stirrer unit, with nominal 20 mL glass sample tubes. Six microcathode oxygen electrodes, mounted in electrode holders, fit into the tubes to measure the oxygen concentration of the test solutions. The electrodes were connected to an interface unit, which converted the current produced by the electrodes into dissolved oxygen readings. The computer software calculated the respiration rate of the six samples, these were compared with the mean control respiration rate to estimate the inhibition caused by each test concentration.
EXPERIMENTAL DESIGN
The test flasks were made up to a total volume of 150 mL by the addition of stock solution (as appropriate), RO water, synthetic sewage and activated sludge.
This test measures the respiration rate of an activated sludge 3 hours after feeding an excess, but standard amount, of a synthetic sewage and compares this with the respiration rate of the same activated sludge in the presence of the test chemical. The reference substance (3,5-DCP) used has known inhibitory effects on respiration and ensures that the batch of sludge used in the test shows a normal level of sensitivity.
For the preliminary test flasks were set up in batches of two or three and aerated at 20 ± 2°C for 3 hours. Each batch included a control flask and either two or three test or reference substance flasks.
For the definitive tests flasks were set up in batches of six and aerated at 20 ± 2°C for 3 hours. Each batch included a control flask and five test or reference substance flasks.
The respiration rate of each flask was measured after 3 hours and compared with the mean respiration rate of the control flask. The rate of oxygen uptake was measured over the linear part of the curve. The rates of oxygen uptake were expressed as mg/L/h.
PRELIMINARY RANGE-FINDER TEST
The preliminary range-finder test included the following vessels, all containing activated sludge at a sludge solids concentration of 1500 mg/L and an excess of synthetic sewage feed:
Duplicate blank controls and single replicates of reference substance, 3,5-DCP, at 1.0, 2.0, 5.0, 10 and 25 mg/L.
Duplicate blank controls, single replicates of Captan Technical at 10 and 100 mg/L and triplicate Captan Technical at 1000 mg/L. Nitrification inhibition vessels: all vessels contained 11.6 mg/L N-allylthiourea (ATU) to inhibit oxidation of ammonia to nitrite. Duplicate blank controls, single replicates of Captan Technical at 10 and 100 mg/L and triplicate Captan Technical at 1000 mg/L.
DEFINITIVE TEST
The definitive study ran in three sets of exposure periods during two days. The test concentrations used for the definitive tests were chosen to cover a range above and below the expected EC50, as determined in the preliminary range-finder test.
The definitive test included the following vessels, all containing activated sludge at a sludge solids concentration of 1500 mg/L and an excess of synthetic sewage feed:
Nominal 0.25, 0.64, 1.6, 4.0 and 10 mg/L concentrations of Captan Technical were prepared in replicates of five. For the reference substance (3,5-DCP), single replicates at 1.0, 2.0, 5.0, 10 and 25 mg/L and a blank control single replicate were prepared.
For the definitive tests, flasks were set up in batches of six and aerated at 20 ± 2°C for 3 hours. Each batch included a control flask and five test or reference substance flasks.
Heterotrophic respiration: Organic carbon oxidation (heterotrophic). Respiration occurring in the presence of the specific nitrification inhibitor- allylthiourea (ATU). ATU is a specific inhibitor of the oxidation of ammonium to nitrite by the first-stage nitrifying bacteria
Nitrification respiration: Rate of oxygen uptake due to nitrification. Ammonium oxidation (nitrification). Calculated by total respiration minus heterotrophic respiration.
The respiration rate of each flask was measured after 3 hours and compared with the mean respiration rate of the control flask. The rate of oxygen uptake was measured over the linear part of the curve. The rates of oxygen uptake were expressed as mg/L/h.

Reference substance (positive control):

yes

Remarks:

3,5-DCP

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

0.64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

8.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid? YES
- Relevant effect levels:
The EC50 of the 3,5-dichlorophenol reference substance should be between 2 and 25 mg/L for total respiration, 5 mg/L to 40 mg/L for heterotrophic respiration and 0.1 mg/L to 10 mg/L for nitrification respiration.
The EC50 for 3,5-dichlorophenol reference substance (calculated using Microsoft Excel) were 3.9, 5.1, 5.0 and 5.2 mg/L for total respiration.

Reported statistics and error estimates:

The EC50 and NOEC were calculated for Captan Technical (total, heterotrophic and nitrification inhibition) together with their 95% confidence intervals, by CETIS (Ref 2) using Linear interpolation (ICPIN).

DEFINITIVE TESTS - RESULTS

The mean total and mean heterotrophic respiration was measured and the difference between them was used to calculate the oxygen uptake rate due to nitrification.

The temperature in all the definitive test flasks were within 20 ± 2°C.

Total Respiration and Inhibition

The results for the definitive total respiration test, including respiration rates, percentage inhibition and pH measurements, are shown in Tables 5 and 6.

The control was compared to the treatments by one-way analysis of variance and Bonferroni adjusted t-tests to identify significant differences (p < 0.05). EC50 was calculated using the linear interpolation (ICPIN) method.

Results based on nominal test concentrations.

3 hour EC50: 8.2 mg/L

95% Confidence Interval: 6.1 - 9.8 mg/L

No Observed Effect Concentration (NOEC): 0.64 mg/L

Table 5 Definitive Total Inhibition Test Batch 1 Results

Test substance	Nominal conc. [mg/L]	pH		Respiration rate (RT) [mg/L/h]		Inhibition (IT) [%]		Statistical significance^3
		Start	End	Individual	Mean (SD)	Individual	Mean (SD)
Control^1	0	7.29	7.89	43.8	-	-	-	-
3,5-DCP	1.0	7.29	8.31	32.4	-	25.9	-	-
	2.0	7.32	8.42	26.8		38.7
	5.0	7.34	8.45	22.1		49.5
	10	7.35	8.40	16.4		62.5
	25	7.35	8.45	8.2		81.3
Control^2	0	7.40	7.95	54.5, 52.7	51.5 (5.6)	-	-	-
Captan Technical	0.25	7.50	7.84	50.1	47.6 (4.1)	2.7	7.5 (7.9)	Non-significant effect
		7.45	7.93	48.6		5.6
		7.42	7.99	52.5		-2.0
		7.43	8.01	44.0		14.5
		7.43	7.85	42.9		16.7
Control^2	0	7.57	7.81	55.8, 49.7	51.5 (5.6)	-	-	-
Captan Technical	0.64	7.54	7.88	44.1	47.3 (7.9)	14.3	8.2 (15.3)	Non-significant effect
		7.53	7.68	38.8		24.6
		7.53	7.87	52.3		-1.6
		7.52	7.86	58.3		-13.2
		7.52	7.96	42.9		16.7
Control^2	0	7.45	7.87	48.5, 48.4	51.5 (5.6)	-	-	-
Captan Technical	1.6	7.42	7.85	41.3	35.3 (6.3)	19.8	31.5 (12.2)	Significant effect
		7.44	7.84	28.7		44.3
		7.43	7.80	40.8		20.7
		7.47	7.98	28.5		44.6
		7.47	7.86	37.0		28.1

¹ The respiration rate for the control run with 3,5-DCP is a mean result of the two reading that were taken for this flask. 3,5-DCP inhibition calculated using the mean respiration rate of the control flask.

² Captan Technical inhibition calculated using mean respiration rates of all control flasks (two readings taken from each flask) run with Captan Technical samples in the definitive total inhibition test (batch 1 and 2).

³ The statistical analysis of respiration rates was performed using the statistical package CETIS Version V1.9.2.6. Significance calculated by Bonferroni Adjusted t Test (α: 5%) using untransformed data with alternative hypothesis C < > T (control response is different to treatment response).

Table 6 Definitive Total Inhibition Test Batch 2 Results

Test substance	Nominal conc. [mg/L]	pH		Respiration rate (RT) [mg/L/h]		Inhibition (IT) [%]		Statistical significance^3
		Start	End	Individual	Mean (SD)	Individual	Mean (SD)
Control^1	0	7.67	7.93	49.0	-	-	-	-
3,5-DCP	1.0	7.85	8.07	34.1	-	30.3	-	-
	2.0	7.79	8.18	29.1		40.6
	5.0	7.75	8.23	24.5		49.9
	10	7.72	8.19	15.3		68.7
	25	7.69	8.34	9.7		80.2
Control^2	0	7.76	7.76	58.8, 58.9	51.5 (5.6)	-	-	-
Captan Technical	4.0	7.83	7.66	44.8	36.1 (7.3)	13.0	29.9 (14.2)	Significant effect
		7.78	7.76	34.7		32.6
		7.81	7.80	40.6		21.1
		7.82	7.84	25.4		50.7
		7.82	7.76	35.0		32.0
Control^2	0	7.63	7.67	43.2, 44.3	51.5 (5.6)	-	-	-
Captan Technical	10	7.85	7.69	19.3	21.6 (2.7)	62.5	58.0 (5.2)	Significant effect
		7.74	7.77	21.7		57.8
		7.82	7.84	22.7		55.9
		7.70	7.88	25.5		50.5
		7.65	7.90	18.9		63.3

¹ The respiration rate for the control run with 3,5-DCP is a mean result of the two reading that were taken for this flask. 3,5-DCP inhibition calculated using the mean respiration rate of the control flask.

² Captan Technical inhibition calculated using mean respiration rates of all control flasks (two readings taken from each flask) run with Captan Technical samples in the definitive total inhibition test (batch 1 and 2).

³ The statistical analysis of respiration rates was performed using the statistical package CETIS Version V1.9.2.6.  Significance calculated by Bonferroni Adjusted t Test (α: 5%) using untransformed data with alternative hypothesis C < > T (control response is different to treatment response).

 

Heterotrophic Respiration and Inhibition

The results for the definitive heterotrophic respiration test, including respiration rates, percentage inhibition and pH measurements, are shown in Table 7.

The control was compared to the treatments by one-way analysis of variance and Dunnett Multiple Comparison Test to identify significant differences (p < 0.05). EC50 was not calculated as none of the test concentrations showed a significant effect.

Results based on nominal test concentrations.

3 hour EC50: > 10 mg/L

No Observed Effect Concentration (NOEC): 10 mg/L

 

Table 7 Definitive Heterotrophic Inhibition Test Results

Test substance	Nominal conc. [mg/L]	pH		Respiration rate (RH) [mg/L/h]		Inhibition (IH) [%]		Statistical significance^4
		Start	End	Individual	Mean (SD)	Individual	Mean (SD)
Control^1	0	7.45	7.34	37.9	-	-	-	-
3,5-DCP	1.0	7.50	7.96	26.2	-	30.9	-	-
	2.0	7.52	8.19	21.6		43.0
	5.0	7.54	8.29	19.1		49.6
	10	7.55	8.30	15.9		58.0
	25	7.55	8.35	11.8		68.9
Control & ATU^2, 3	0	7.56	8.45	12.4	9.7 (3.5)	-	-	-
Captan Technical and 11.6 mg/L ATU^2	0.25	7.53	8.54	13.1	15.1 (1.9)	-35.6	-55.9 (19.3)	Non-significant effect
		7.54	8.53	13.7		-41.8
		7.55	8.56	15.9		-64.6
		7.56	8.58	14.8		-53.2
		7.58	8.52	17.8		-84.3
Control & ATU^2, 3	0	7.45	8.40	11.5	9.7 (3.5)	-	-	-
Captan Technical and 11.6 mg/L ATU^2	0.64	7.52	8.52	16.4	15.3 (1.9)	-69.8	-58.0 (19.3)	Non-significant effect
		7.54	8.54	14.1		-46.0
		7.56	8.59	17.4		-80.1
		7.57	8.59	12.7		-31.5
		7.58	8.61	15.7		-62.5
Control & ATU^2, 3	0	7.44	8.48	12.6	9.7 (3.5)	-	-	--
Captan Technical and 11.6 mg/L ATU^2	1.6	7.43	8.50	Excluded as outlier	7.2 (1.0)	Excluded as outlier	26.0 (10.2)	Non-significant effect
		7.45	8.50	6.2		35.8
		7.47	8.51	8.1		16.1
		7.48	8.49	7.9		18.2
		7.49	8.52	6.4		33.7
Control & ATU^2, 3	0	7.30	8.30	6.5	9.7 (3.5)	-	-	-
Captan Technical and 11.6 mg/L ATU^2	4.0	7.25	8.35	5.5	9.6 (3.4)	43.1	0.8 (35.0)	Non-significant effect
		7.43	8.41	6.5		32.7
		7.35	8.48	12.4		-28.4
		7.47	8.44	10.7		-10.8
		7.56	8.47	12.8		-32.5
Control & ATU^2, 3	0	7.22	8.11	5.3	9.7 (3.5)	-	-	-
Captan Technical and 11.6 mg/L ATU^2	10	7.29	8.21	6.9	6.1 (2.7)	28.6	36.9 (27.7)	Non-significant effect
		7.34	8.21	4.5		53.4
		7.43	8.26	7.9		18.2
		7.41	8.24	2.3		76.2
		7.39	8.28	8.9		7.9

3,5-DCP inhibition calculated using the respiration rate of this control flask.

² Flasks had 11.6 mg/L ATU added to inhibit nitrification

³ Captan Technical and ATU inhibition calculated using mean respiration rates of all control and ATU flasks run with Captan Technical and ATU samples in the definitive heterotrophic inhibition test.

⁴ The statistical analysis of respiration rates was performed using the statistical package CETIS Version V1.9.2.6.  Significance calculated by Dunnett Multiple Comparison Test (α: 5%) using untransformed data with alternative hypothesis C > T (control response greater than treatment response).

Nitrification Respiration and Inhibition

The calculated results for the definitive nitrification respiration test, are shown in Table 8

The control was compared to the treatments by one-way analysis of variance and Dunnett Multiple Comparison Test to identify significant differences (p < 0.05). EC50 was calculated using the linear interpolation (ICPIN) method.

EC50 was calculated using the linear interpolation (ICPIN) method.

Results based on nominal test concentrations.

3 hour EC50: 7.0 mg/L

95% Confidence Interval: 1.8 - 9.4

No Observed Effect Concentration (NOEC): 0.64

Table 8 Nitrification Inhibition Results

Test substance	Nominal conc. [mg/L]	Respiration rate^2 (RH) [mg/L/h]		Inhibition (IH) [%]		Statistical significance^3
		Individual	Mean (SD)	Individual	Mean (SD)
Nitrification Control^1	0	41.2	41.8 (6.3)	-	-	-
Nitrification respiration Captan Technical	0.25	35.0	32.6 (4.1)	16.2	22.1 (9.7)	Non-significant effect
		33.5		19.8
		37.4		10.5
		28.9		30.8
		27.8		33.4
Nitrification Control^1	0	41.3	41.8 (6.3)	-	-	-
Nitrification respiration Captan Technical	0.64	28.8	32.0 (7.9)	31.0	23.4 (18.8)	Non-significant effect
		23.5		43.7
		37.0		11.4
		43.0		-2.9
		27.6		33.9
Nitrification Control^1	0	35.9	41.8 (6.3)	-	-	--
Nitrification respiration Captan Technical	1.6	34.2	28.1 (6.3)	18.3	32.8 (15.1)	Significant effect
		21.6		48.5
		33.7		19.5
		21.4		48.9
		29.9		28.6
Nitrification Control^1	0	52.4	41.8 (6.3)	-	-	-
Nitrification respiration Captan Technical	4.0	35.2	26.5 (7.3)	15.8	36.6 (17.5)	Significant effect
		25.1		39.9
		31.0		25.8
		15.8		62.2
		25.4		39.2
Nitrification Control^1	0	38.5	41.8 (6.3)	-	-	-
Nitrification respiration Captan Technical	10	13.2	15.5 (2.7)	68.4	62.9 (6.4)	Significant effect
		15.6		62.7
		16.6		60.3
		19.4		53.6
		12.8		69.4

 

The statistical analysis of respiration rates was performed using the statistical package CETIS Version V1.9.2.6.

¹ Nitrification inhibition was calculated using the average of all the control replicates. 

² Nitrification respiration was calculated using individual total respiration - mean heterotrophic respiration.

³ The statistical analysis of respiration rates was performed using the statistical package CETIS Version V1.9.2.6.  Significance calculated by Dunnett Multiple Comparison Test (α: 5%) using untransformed data with alternative hypothesis C < > T (control response is different to treatment response).

Validity Criteria

The validity requirements of the OECD guideline state:

• The respiration rate of control vessels should be at least 20 mg O2/g activated sludge/h;


• The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of the test;


• The EC50 of the 3,5-dichlorophenol reference substance should be between 2 and 25 mg/L for total respiration, 5 mg/L to 40 mg/L for heterotrophic respiration and 0.1 mg/L to 10 mg/L for nitrification respiration.

The results for the control flasks validity criteria are given in Table 9. The respiration rate of the all the control vessels ranged from 25.3 to 39.3 mg O2/g activated sludge/h. The coefficient of variation of oxygen uptake rate in the control replicates was 12.4% at the end of the test.

The EC50 for 3,5-dichlorophenol reference substance (calculated using Microsoft Excel) were 3.9, 5.1, 5.0 and 5.2 mg/L for total respiration (Table 10).

Table 9 Control Flasks Validity Criteria

Flask no.	Respiration rate RT (mg/L/h)	Respiration rate RS (mg/g/h)
Definitive test 1	45.4	30.3
Definitive test 5	42.1	28.1
Definitive test 7	54.5	36.3
Definitive test 7	52.7	35.1
Definitive test 13	55.8	37.2
Definitive test 13	49.7	33.1
Definitive test 19	48.5	32.3
Definitive test 19	48.4	32.3
Definitive test 25	49.4	32.9
Definitive test 25	48.5	32.3
Definitive test 31	58.8	39.2
Definitive test 31	58.9	39.3
Definitive test 37	43.2	28.8
Definitive test 37	44.3	29.5
Definitive test 1a	37.9	25.3
Definitive test 7a^1	12.4	8.3
Definitive test 13a^1	11.5	7.7
Definitive test 19a^1	12.6	8.4
Definitive test 31a^1	6.5	4.3
Definitive test 37a^1	5.3	3.5
Mean control respiration rate (Rs) (mg/g/h)		32.8
Coefficient of variation (CV) (%)		12.4

1 These controls flasks had 11.6 mg/L ATU added to inhibit nitrification.
These are not included in the mean and CV calculations

Table 10 Reference Substance Validity Criteria

3,5-DCP Reference substance run with:

Total respiration EC50 (mg/L)

Validity criteria fulfilled:

yes

Conclusions:

The mean total and mean heterotrophic respiration were measured and the difference between them was used to calculate the oxygen uptake rate due to nitrification.
For total respiration, the 3-hour EC50 was determined to be 8.2 mg/L, the NOEC was determined as 0.64 mg/L.
For heterotrophic respiration, the 3-hour EC50 was determined to be > 10 mg/L, the NOEC was determined as 10 mg/L. For nitrification respiration, the 3-hour EC50 was determined to be 7.0 mg/L, the NOEC was determined as 0.64 mg/L.

Executive summary:

The inhibitory effect of Captan Technical on the respiration rate of activated sludge micro-organisms, typical of those found in domestic sewage treatment, was determined in a respirometric test. The test comprised total inhibition, heterotrophic and nitrification inhibition.

A range-finding test was carried out with concentrations of 10, 100 and 1000 mg/L. Based on the results of the preliminary test the main study was carried out with test concentrations of 0.25, 0.64, 1.6, 4.0 and 10 mg/L. Five replicate for each concentration and untreated controls were included in the test. No vehicle was used. 3,5-dichlorophenol (3,5-DCP) was used as reference item at test concentrations of 1.0, 2.0, 5.0, 10 and 25 mg/L.

The inhibitory effects of the test and reference item were determined in comparison to the control respiration rates.

The mean total and mean heterotrophic respiration were measured and the difference between them was used to calculate the oxygen uptake rate due to nitrification.
For total respiration, the 3-hour EC50 was determined to be 8.2 mg/L, the NOEC was determined as 0.64 mg/L.
For heterotrophic respiration, the 3-hour EC50 was determined to be > 10 mg/L, the NOEC was determined as 10 mg/L. For nitrification respiration, the 3-hour EC50 was determined to be 7.0 mg/L, the NOEC was determined as 0.64 mg/L.

All validity criteria of the test guideline were met.

Description of key information

Toxicity to microorganisms, Respiration Inhibition Test (3h, static conditions), activated sludge (municipal treatment plant), OECD No. 209, total respiration: NOEC: 0.64 mg/L EC50: 8.2 mg/L; heterotrophic respiration: EC₅₀ = > 10 mg/L, NOEC = 10 mg/L; nitrification respiration: EC₅₀ = 7.0 mg/L, NOEC = 0.64 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

8.2 mg/L

EC10 or NOEC for microorganisms:

0.64 mg/L