N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid

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:

2017-11-24 to 2018-02-21

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:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was weight out directly in the Erlenmeyer flasks
- Controls: Reference item for total respiration: Copper (ll) sulphate pentahydrate p.a.; Reference item for Nitrification: N-Methylaniline also directly weight out in the Erlenmeyer flasks.

Test organisms (species):

activated sludge

Details on inoculum:

- Pretreatment: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L s 10 %. The sludge was used within 24 h after sampling.
- Initial biomass concentration: 2.98 g/L, corresponding to 1.49 g/L in the test vessel for replicates without ATU and 2.83 g/L, corresponding to 1.43 g/L in the test vessel for replicates with ATU (N-allylthiourea).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.1 °C

pH:

The pH value of the activated sludge was determined prior to test start. The pH value of the synthetic waste water was determined prior to use. Adjustment to 7.5 ± 0.5 was not necessary.

Dissolved oxygen:

above 60 - 70 % saturation

Nominal and measured concentrations:

10, 32, 100, 320, 1000 mg a.i../L (corresponding to 11.1, 35.6, 111, 356, 1111 mg test item/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks, DIN 12380
- Aeration: Permanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): Triplicates at the beginning of the test + triplicates at the end of the replicates without ATU (N-allylthiourea). Triplicates at the beginning of the replicates with ATU (N-allylthiourea) + triplicates at the end of the test
- Sludge concentration (weight of dry solids per volume): 3.0 g/L ±10 %
- Nutrients provided for bacteria: Synthetic waste water according to OECD Guideline 209
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea (ATU)

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration Inhibition Test with activated sludge to determine the inhibition of total, heterotrophic and ammonium respiration in %.
TEST CONCENTRATIONS
- Range finding study
- Test concentrations: In the preliminary test, additional replicates were prepared to determine the inhibition of nitrification. One replicate of 10, 100, 1000 mg/L were measured with addition of N-allylthiourea (ATU). A second preliminary test (non GLP) was carried out with the concentrations of the test item Tetrahydrofolic acid of 500 - 1000 mg/L with one replicate each and one replicate each with pH adjustment to determine the pH effect and the inhibition of nitrification.
- Results used to determine the conditions for the definitive study: Based on the results of the preliminary test the definitive study was carried out with 5 concentrations within the range of 10 to 1000 mg a.i./L (with and without ATU) in a geometrical series and with a dilution factor of 3.2.

Reference substance (positive control):

yes

Remarks:

Reference item for total respiration: Copper (ll) sulphate pentahydrate p.a.; Reference item for Nitrification: N-Methylaniline

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: Due to an inhibition of 7% at 100 mg/L and lack of biological relevance, 100 mg/L should be regarded as NOEC

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

146 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

671 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of nitrification rate

Remarks on result:

other: Due to an inhibition of 7% at 100 mg/L and lack of biological relevance, 100 mg/L should be regarded as NOEC

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

39 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of nitrification rate

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

246 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of nitrification rate

Details on results:

The NOEC of the total respiration was found to be 32 mg/L. At 100 mg a.i./L, 7% inhibition was observed. Although this inhibition was found to be statistically significant, this inhibition might not be considered to be biologically relevant. Accordingly, when factoring in the biological relevance of an inhibition of only 7%, the statistically significant result should be considered an artefact. Therefore, 100 mg/L should be considered as NOEC.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: The EC50 of the reference item copper (ll) sulphate pentahydrate was 116 mg/L and is in the range of 53 - 155 mg/L.

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculation. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-value for both tests was 0.05. The α-value (acceptable probability of incorrectly concluding that there is a difference) is α = 0.05. The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression (biphasic regression where necessary) and linear regression, respectively, using software GraphPadPrism. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.

Table 6: Oxygen Uptake Rates and Specific Respiration Rates of the total Respiration of the Control

	Repl.	Oxygen Concentration* [mg O2/L]	Oxygen uptake Rate R [mg O2/(L x h)]	Specific Respiration rate Rs [mg O2/g x h]
Control	1	7.27	34.2	23.0
	2	6.90	34.0	22.8
	3	6.50	34.5	23.2
	4	7.58	33.0	22.1
	5	7.67	31.6	21.2
	6	7.49	32.8	22.0
	Mean value		33.4	22.4
	Standard deviation		1.0
	CV [%]		3.0

Table 7: Oxygen Uptake Rates and Inhibition of the total Respiration of the Test item Concentrations

Test item concentration [mg a.i./L]	Repl.	Oxygen Concentration* [mg O2/L]	Oxygen uptake Rate R [mg O2/(L x h)]	Inhibition [%]	Mean Inhibition [%]
10	1	6.43	33.8	-1	2
	2	6.51	33.5	0
	3	6.88	34.2	-2
	4	7.12	30.1	10
	5	6.65	33.0	1
32	1	7.17	33.4	0	-2
	2	6.31	33.5	0
	3	6.34	34.8	-4
	4	6.47	33.4	0
	5	6.47	35.3	-6
100	1	7.18	31.2	7	7
	2	6.68	29.4	12
	3	6.96	31.3	6
	4	6.63	30.1	10
	5	6.79	33.1	1
320	1	7.21	26.3	21	25
	2	7.44	23.8	29
	3	7.06	25.4	24
	4	7.18	25.5	24
	5	7.01	24.2	28
1000	1	7.67	10.9	67	65
	2	7.89	11.1	67
	3	7.76	12.4	63
	4	7.51	11.7	65
	5	7.99	11.9	64

Table 8: Oxygen Uptake Rates of the heterotrophic Respiration of the Control Replicates with ATU

	Repl.	Oxygen Concentration* [mg O2/L]	Oxygen uptake Rate R [mg O2/(L x h)]
Control	1	8.09	12.9
	2	8.13	11.7
	3	8.15	12.9
	4	7.91	13.9
	5	7.85	13.1
	6	8.15	12.3
	Mean value		12.8
	Standard deviation		0.7
	CV [%]		5.3

Table 9: Oxygen Uptake Rates and lnhibition of the heterotrophic Respiration of the Test Item Concentrations

Test Concentration [mg a.i./L]		Repl.	Oxygen Concentration* [mg O2/L]	Oxygen uptake Rate R [mg O2/(L x h)]	Inhibition [%]	Mean Inhibition [%]
Test item	10 + ATU	1	8.08	12.9	-1	-12
		2	7.74	15.0	-17
		3	7.77	14.9	-16
		4	7.59	14.1	-10
		5	7.58	15.0	-17
	32 + ATU	1	8.22	13.8	-8	-10
		2	8.18	14.1	-10
		3	8.33	14.2	-11
		4	7.96	14.4	-13
		5	8.38	13.8	-8
	100 + ATU	1	8.00	16.6	-30	-20
		2	7.60	15.0	-17
		3	7.47	15.8	-23
		4	7.15	14.3	-12
		5	7.12	15.3	-20
	320 + ATU	1	7.97	16.0	-25	-32
		2	7.42	17.3	-35
		3	7.44	17.3	-35
		4	7.33	16.8	-31
		5	7.88	17.4	-36
	1000 + ATU	1	7.66	11.8	8	0
		2	7.69	12.7	1
		3	7.57	14.6	-14
		4	7.43	11.4	11
		5	7.49	13.3	-4

*) determined at start of the measurement

Table 10: Calculated Oxygen Uptake Rates of the Nitrification of the Control

	Repl.	Oxygen Concentration* [mg O2/L]
Control	1	21.3
	2	22.3
	3	21.6
	4	19.1
	5	18.5
	6	20.5
Mean value		20.6
Standard deviation		1.4
CV [%]		6.6

Table 11: Calculated Oxygen Uptake Rates and Inhibition of the Nitrification of the Test Item Concentrations

Test Concentration [mg a.i./L]		Repl.	Oxygen uptake Rate R [mg O2/(L x h)]	Inhibition [%]	Mean Inhibition [%]
Test item	10	1	20.9	-1	10
		2	18.5	10
		3	19.3	6
		4	16.0	22
		5	18.0	13
	32	1	19.6	5	3
		2	19.4	6
		3	20.6	0
		4	19.0	8
		5	21.5	-4
	100	1	14.6	29	24
		2	14.4	30
		3	15.5	25
		4	15.8	23
		5	17.8	14
	320	1	10.3	50	61
		2	6.5	68
		3	8.1	61
		4	8.7	58
		5	6.8	67
	1000	1	-0.9	104	106
		2	-1.6	108
		3	-2.2	111
		4	0.3	99
		5	-1.4	107

Validity criteria fulfilled:

yes

Conclusions:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Tetrahydrofolic acid. The test was carried out under static conditions with the test item concentrations 10 - 32 - 100 -320 - 1000 mg a.i./L with and without ATU.
The NOEC of Tetrahydrofolic acid for total respiration and nitrification is 32 mg a.i./L.
The EC10 for total respiration is 146 mg a.i./L and the EC50 is 671 mg a.i./L. ,
The NOEC for heterotrophic respiration is > 1000 mg a.i./L.
The EC10 and EC50 for heterotrophic respiration are >1000 mg a.i./L.
The EC10 for nitrification is 39.0 mg a.i./L and the EC50 is 246 mg a.i./L.

Description of key information

- OECD 209 guideline study, GLP, respiration inhibition test with activated non-adapted sludge, 3h exposure to 10, 32, 100, 320 and 1000 mg a.i./L of the test item; NOEC for total respiration and nitrification is 32 mg a.i../L (100 mg/L should be considered as NOEC for total respiration). EC10 for total respiration = 146 mg a.i./L; EC50 = 671 mg a.i./L. ,NOEC for heterotrophic respiration = > 1000 mg a.i./L. The EC10 and EC50 for heterotrophic respiration = >1000 mg a.i./L. The EC10 for nitrification = 39.0 mg a.i./L and the EC50 = 246 mg a.i./L.

Key value for chemical safety assessment

EC50 for microorganisms:

671 mg/L

EC10 or NOEC for microorganisms:

146 mg/L

Additional information

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Tetrahydrofolic acid. The test was carried out under static conditions with the test item concentrations 10 - 32 - 100 -320 - 1000 mg a.i./L with and without ATU. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of total respiration for the test item replicates ranged from - 2 % to 65 %. The heterotrophic respiration was not inhibited. The mean inhibition of nitrification for the test item replicates ranged from 3 % to 106 %.

The NOEC of the total respiration was found to be 32 mg/L. At 100 mg/L test item, 7% inhibition was observed. Although this inhibition was found to be statistically significant, this inhibition might not be considered to be biologically relevant. Accordingly, when factoring in the biological relevance of an inhibition of only 7%, the statistically significant result should be considered an artefact. Therefore, 100 mg/L should be considered as NOEC.

The NOEC of Tetrahydrofolic acid for total respiration and nitrification is 32 mg a.i./L

(100 mg/L should be considered as NOEC for total respiration, for details see text).

The EC10 for total respiration is 146 mg a.i./L and the EC50 is 671 mg a.i./L. ,

The NOEC for heterotrophic respiration is > 1000 mg a.i./L.

The EC10 and EC50 for heterotrophic respiration are >1000 mg a.i./L.

The EC10 for nitrification is 39.0 mg a.i./L and the EC50 is 246 mg a.i./L.