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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:

2015-04-09

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

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1000 mg/L was weighed out directly in Erlenmeyer flasks and stirred overnight
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 1000 mg/L
- Controls: Test medium without test or reference item. Triplicates at the beginning of the test + triplicates at the end of the test
-Reference Item: three concentrations within the range 58 - 180 mg/L and a dilution factor of 1.8, Triplicates

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Non-adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 3.06 g/L, corresponding to 1.53 g/L in the test vessel

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

20.9 °C

pH:

pH-value of the activated sludge 7.87
pH-value of the synthetic waste water 7.38

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, 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): 6 (Triplicates at the beginning of the test + triplicates at the end of the test.)
- Composition of test medium:

Table for the Test Item Concentrations
Test concentration [mg/L] 1000
Test Item [mg] 500
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250


Dilution Table for the Reference Item
Reference Item Reference concentration [mg/L]
58 100 180
Stock solution [µL] * 96.7 166.7 300
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250
The reference item con. were diluted out of a stock solution
*) concentration of stock solution 300 g/L



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
Inhibition of Respiration (Preliminary Tests)

Inhibitions in the Preliminary Tests

Nominal Test Item Concentration
[mg/L] pH

(test item in demin. water) pH

(adjusted to) pH

(test item in medium) Inhibition


[%]
1st preliminary test – with pH adjustment, direct aeration
10 6.85 - 7.22 -8
5.55 - 7.28 -10
100 5.51 6.90* 7.26 -3
6.52 - 7.28 -7
1000 5.64 7.47* 7.33 -12
5.57 - 7.35 -10
2nd preliminary test – without pH adjustment, stirred overnight
10 6.24 - 7.30 0
6.25 - 7.28 8
100 6.33 - 7.29 3
6.53 - 7.35 4
1000 7.73 - 7.44 -1
6.44 - 7.42 -4
*) pH adjusted with 0.5 mol/L NaOH

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: NOEC: ≥ 1000 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) and DUNNETT’s test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression and linear regression, respectively, using software GraphPadPrism. Calculations of the confidence intervals for the EC-values were carried out using standard procedures.

From the oxygen depletion the oxygen uptake rates were calculated in mg O₂/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O₂/g×h.

The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison with the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration of Control, Test and Reference Item Concentrations

Test and Reference Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	36.0	-	-
	2	40.0
	3	35.2
	4	40.4
	5	38.0
	6	37.6
Test item 1000	1	36.8	3	-2
	2	40.0	-6
	3	38.8	-2
Reference item 58	1	27.2	28	29
	2	25.6	32
	3	27.2	28
100	1	19.6	48	48
	2	19.2	49
	3	19.6	48
180	1	10.0	74	74
	2	9.6	75
	3	10.0	74

Repl. = Replicate

Coefficient of variation of oxygen uptake rates of the control: 5.5%

Suspended solids in the test vessel: 1.53 g/L

Specific Respiration Rates of the Control Replicates

		Repl.	Specific Respiration Rate Rs [mg O2/g×h]
	Control	1	23.5
		2	26.1
		3	23.0
		4	26.4
		5	24.8
		6	24.6
Mean value			24.7

Repl. = Replicate

Suspended solids in the test vessel: 1.53 g/L

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item is ≥ 1000 mg/L. The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out with the test item from 2015-04-08 to 2015-04-09 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the limit test item concentration 1000 mg/L. 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 respiration for the test item replicates was -2 %.

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 99.8 mg/L. The validity criteria of the test guideline were fulfilled.

 

NOEC of test item

		Test item Concentration [mg/L]
NOEC*		≥ 1000

*) No statistically significant effect (P = 0.645), ANOVA

 

 

The NOEC of the test item is ≥ 1000 mg/L. LICOWAX R 21 S FL caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L.

Description of key information

The NOEC of the test item is ≥ 1000 mg/L. The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L