[No public or meaningful name is available]

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:

2018-10-10 to 2018-10-11

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

Specific details on test material used for the study:

Test item Licocare RBW 300
Batch number DEF2114527
CAS No. 1850357-57-1
Active ingredient Licocare RBW 300 FL TP
Chemical name Wax, rice bran, oxidized, calcium salts
Purity (certified) 99.75 % w/w
Appearance yellow solid flakes
Water solubility < 1g/L (OECD 105)
Expiry date 2020-11-05
Recommended storage Room temperature

The test item and the information concerning the test item were provided by the sponsor.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1000 mg/L were weighed out directly in Erlenmeyer flasks.
- 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 concentration 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)
- Receipt: 2018-10-10
- 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: 2.95 g/L (corresponding to 1.48 g/L in the test vessels)

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 7.45
pH-value of the synthetic waste water 7.21

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 3
- 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.
- Pretreatment: grinding in mortar, stirring overnight

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 (Triplicates at the beginning of the test + triplicates at the end of the test)
-No. of vessels per reference item (replicates): 9 (three concentrations within the range 58 - 180 mg/L and a dilution factor 1.8)
- Composition of test medium:

Test concentration [mg/L] 1 000
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 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
* Stock solution with 300g/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 every 3 seconds after transferring the solution to the measuring chamber and the oxygen depletion was recorded for at least 5 minutes with the software PreSens Measurement Studio 2.

TEST CONCENTRATIONS

Inhibitions in the Preliminary Tests
A preliminary range finding test (non GLP) was carried out with the concentrations of the test item Licocare RBW 300 of 10 – 100 – 1000 mg/L with one replicate each. The pH was measured in one replicate with 1000 mg/L test item. The flasks were aerated for 3 hours.

In the preliminary test, additional replicates were prepared to determine the inhibition of nitrification. One replicate each of 10 – 100 – 1000 mg/L were measured with addition of Allylthiourea (ATU).

Inhibitions in the Preliminary Tests

Nominal Test Item Concentration
[mg/L] pH
(test item in medium) Inhibition of total respiration (without ATU)
[%] Inhibition of heterotrophic respiration (with ATU)
[%] Inhibition of nitrification
[%]
10 - 3 1 5
100 - 6 5 7
1000 7.58 3 -4 7



Based on the results of the preliminary test it was concluded that the heterotrophic respiration as well as the nitrification are not inhibited. Therefore, the definitive study was carried out with the concentration of 1000 mg/L in 3 replicates (limit test to determine the NOEC). The test item was ground in a mortar, weighed out directly in the Erlenmeyer flasks and stirred overnight.

Triplicates of the control without test item were included at the beginning and at the end of the test.

The reference item was tested with three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of approx. 1.8.

A fresh sample of activated sludge taken from the sewage plant Hildesheim was used for the test procedure. It was washed twice with a dry sludge concentration of 3.0 g/L ± 10 %.

The oxygen uptake rates of all samples were determined following an incubation period of 3 h.

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Key result

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

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 P-value (acceptable probability of incorrectly concluding that there is a difference) is P=0.05.

The EC-value of the reference item was calculated by linear regression using software GraphPad Prism. 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.

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 the Test Item Concentration

Test Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
1000	1	7.64	24.9	-2	-2
	2	7.36	25.6	-5
	3	7.00	23.7	2

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

Reference Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
58	1	7.25	17.3	29	31
	2	6.96	16.3	33
	3	6.23	16.5	32
100	1	7.07	13.3	45	44
	2	7.34	14.0	42
	3	6.77	13.3	45
180	1	7.78	5.1	79	79
	2	7.37	5.5	77
	3	6.87	4.8	80

Repl. = Replicate        

Specific Respiration Rates of the Control Replicates

	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Specific Respiration Rate Rs
		[mg O2/L]	[mg O2/(L×h)]	[mg O2/g×h]
Control	1	6.86	23.4	15.8
	2	7.35	24.6	16.6
	3	7.13	23.6	15.9
	4	7.42	25.7	17.4
	5	7.24	25.1	17.0
	6	6.41	23.1	15.6
	Mean value		24.3	16.4
	Standard deviation		0.95
	CV [%]		3.91

Repl. = Replicate

 

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item Licocare RBW 300 is ≥ 1000 mg/L.
Licocare RBW 300 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 Licocare RBW 300 on 2018-10-10 to 2018-10-11, with the definitive exposure phase on 2018-10-11 at the test facility. 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 98.3 mg/L. The validity criteria of the test guideline were fulfilled.

NOEC of Licocare RBW 300

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

               *) No statistically significant effect (P = 0.524), ANOVA

                    

The NOEC of the test item Licocare RBW 300 is ≥ 1000 mg/L.

Licocare RBW 300 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

Study 1

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out with the test item Licocare RBW 300

The NOEC of the test item Licocare RBW 300 is ≥ 1000 mg/L.

Licocare RBW 300 caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L.

 

Study 2

Under the present test conditions, 3h-EC50 of the test substance for the total oxygen uptake was higher than 1001 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L