6-Amino-5-chloro-2-cyclopyrimidine-4-carboxylic 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

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Pretreatment (of test without ATU): test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours. The pH (3.7 - 3.8) was adjusted to pH 7.1 - 7.5 with NaOH. For the reference compound a stock solution at a concentration of 500 mg/L was prepared by dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluting to 0.5 litre with deionised water. The pH was adjusted to pH 7 - 8 with HCl.
- Pretreatment (of test with ATU): test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours. The pH (3.7 - 3.8) was adjusted to pH 7.2 - 7.3 with NaOH. For the ATU-solution 2.32 g N-allylthiourea were diluted with deionized water to 1 litre. 1.25 mL of the solution were given to all replicates for the determination of the heterotrophic oxidation immediately before start of the incubation period.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Municipal WWTP Odenthal , Germany
- Method of cultivation: aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
- Preparation of inoculum for exposure: sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 3500 rpm and 20°C) the supernatantwas decanted again. The calculated amount of sludge was dissolved in
synthetic medium and then filled up to a defined end volume with deionised water.
- Initial biomass concentration: 3 g/L dw suspended solids.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

18.6 - 19.2°C

pH:

7.9 - 8.6

Nominal and measured concentrations:

Nominal test substance concentrations: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL glass Erlenmeyer flasks
- Type: closed
- Fill volume: 250 mL
- Aeration: yes (50-100 L/h with clean oil-free air)
- No. of vessels per concentration (replicates): 3 replicates without ATU, 2 replicates with ATU
- No. of vessels per control (replicates): 6 replicates without ATU, 4 replicates with ATU
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration: 1000 mg/L suspended solids
- Nutrients provided for bacteria: synthetic waste water feed
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Details on termination of incubation: content of the Erlenmeyer flasks was completely transferred to 250 mL BOD bottles
EFFECT PARAMETERS MEASURED: oxygen content (after 3 h)

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol: 2.5, 5, 10, 20 and 40 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 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: EC50: 12.801 mg/L

Reported statistics and error estimates:

STUDENT-t test for Homogeneous Variances: Pair-wise comparison of treatments with "Control" by the t test procedure. Significance was Alpha = 0.05. onesided smaller; the differences are significant in case p(i) <= Alpha ; p(F): probability of F computed by a F-test (Ho: var1 = var2 (homogeneity); p(F) > 0.05 is the criterion of variance homogeneity. (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).

Table 1: Total respiration (without ATU) rates after 3 hours incubation period, percentage inhibition, temperature and pH values

Treatment [mg/L]		Respiration rate	Mean Temp.	pH-	Inhibition
		[mg/L · h]	[°C]	value	[%]
Control 1	--	38.167	19.7	8.4	--
Control 2	--	34.138	19.1	8.5	--
Control 3	--	34.337	19.2	8.4	--
Control 4	--	34.694	19.0	8.5	--
Control 5	--	35.333	18.9	8.5	--
Control 6	--	30.213	19.0	8.6	--
Control, mean		34.480	--	--	--
Test item	100	36.233	18.9	8.6	0.000
Test item	100	33.893	18.6	8.6	1.705
Test item	100	34.428	18.7	8.6	0.151
Test item, mean	100	34.851	--	--	0.619
Physico-chemical oxygen consumption control	100	0.233	18.6	7.9	--
Reference compound	2.5	33.535	19.0	8.5	2.742
Reference compound	5	34.710	19.2	8.5	0.000
Reference compound	10	18.460	19.1	8.5	46.461
Reference compound	20	9.574	18.6	8.5	72.232
Reference compound	40	5.766	18.8	8.5	83.277

Table 2: Heterotrophic respiration (with ATU) rates after 3 hours incubation period, percentage inhibition, temperature and pH values

Treatment [mg/L]		Respiration rate	Mean Temp.	pH-	Inhibition
		[mg/L · h]	[°C]	value	[%]
Control 1		31.534	19.0	8.5	--
Control 2		34.068	19.0	8.5	--
Control 3		35.289	18.9	8.6	--
Control 4		37.250	19.1	8.6	--
Control, mean		34.535	--	--	--
Test item	100	32.693	18.9	8.6	5.336
Test item	100	32.895	19.0	8.6	4.749
Test item, mean	100	32.794	--	--	5.043

Table 3: Validity criteria for OECD 209.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.	34.480 mg oxygen per one gram of activated sludge (dry weight of suspended solids)	yes
The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.	7.4 %	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

NOEC (3 h) ≥ 100 mg/L (nominal, OECD 209, activated sludge respiration rate)

EC50 (3 h) > 100 mg/L (nominal, OECD 209, activated sludge respiration rate)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

One GLP study on the toxicity of the test substance to microorganisms is available.

This key study was performed according to OECD guideline 209 with activated sludge from domestic sewage treatment plant. The activated sludge was exposed to the test substance at a limit test item concentration of 100 mg/L. To determine the heterotrophic oxidation, additional controls and exposure groups (test item concentration of 100 mg/L), all containing 1.25 mL of specific nitrification inhibitor, ATU-solution (N-allylthiourea), were tested.

The respiration rate of each mixture was determined after aeration periods of 3 hours. As no significant inhibitory effect was measured at a limit test item concentration of 100 mg/L no statistical analysis was required to determine the EC50. The NOEC (3 h) was calculated to be the test concentration of 100 mg/L.