Polar modified Rice Bran Wax

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

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:

other: The national hazardous chemical management standardization technical committee. GB/T 21796-2008 Chemicals- Activated Sludge, Respiration Inhibition Test[S]. Beijing: China Standards Press.2008.

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals Effects on Biotic Systems, Activated Sludge, Respiration Inhibition Test [M]. Second Edition. Beijing: China Environmental Science Press. 2013: 73 - 85.

Version / remarks:

2013

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Version / remarks:

1996

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

2010

GLP compliance:

yes

Analytical monitoring:

no

Test organisms (species):

activated sludge

Details on inoculum:

Name Activated sludge
Source Near the exit of the aeration tank from Water Purification Plant
Batch No. IAs20210517-1
Selection reason The inoculum is recommended by "The Guidelines for the Testing of Chemicals"
Treatment After collection, any coarse particles and impurities were removed from the sludge by sieving of a strainer with the aperture of 80 meshes, then washed and re-suspended in chlorine free tap water. This was cultivated for one day by 50 mL of synthetic sewage feed per litre of activated sludge was fed and aerated under the test temperature of 20°C±2°C. Before the study, the solids dry weight of the activated sludge suspension was determined, followed by adjusting the initial concentration of the suspended solids dry weight to 3.0 g/L with the chlorine free tap water, and adjusting its pH value to 7.29 before being used.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19.4 °C - 21.7 °C

pH:

7.08 - 7.18

Details on test conditions:

Test substance LICOCARE RBW 106
Test group Total oxygen uptake (FT1-5)
Abiotic control (FA1-3)
Reference mixtures (FR1-5, FHR1-5)
Blank controls (FTB1-4, FHB1-2)
Test substance final concentrations 10 mg/L, 100 mg/L, 1000 mg/L
Replication Triplicates at the highest tested concentration
(1000 mg/L), one replicates at the other
groups.
Activated sludge final concentration 1.5 g/L
Final volume 500 mL
Test temperature 19.4°C - 21.7°C (within 20°C±2°C)
Test duration 3 h
Test condition Aeration and agitation

Reference substance (positive control):

yes

Remarks:

3,5-diclorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Validity criteria fulfilled:

yes

Conclusions:

Under the present test conditions, 3h-EC50 of the test substance for the total oxygen uptake was higher than 1000 mg/L.

Executive summary:

(1) The sensitivity test of the activated sludge

The average of specific oxygen uptake rates of the blank controls (without ATU) was    25.9 mg/(g·h), which was greater than 20 mg/(g·h). The coefficients of variation of the blank controls were within 1.6% - 4.2%, which were less than 30% and met the validity of the test.

The 3h-EC₅₀ of 3,5-DCP for total oxygen uptake was 7.6 mg/L, which was within the range of 2 mg/L to 25 mg/L. 3h-EC₅₀ for heterotrophic respiration was 14.1 mg/L, which was within the range of 5 mg/L to 40 mg/L. 3h-EC₅₀ for nitrification respiration was 0.3 mg/L, which was within the range of 0.1 mg/L to 10 mg/L. All of these results revealed that the sensitivity of the activated sludge used in the test could meet the requirement of the test.

(2) Range-finding test

The average of specific oxygen uptake rates of the blank controls (without ATU) was    28.3 mg/(g·h), which was greater than 20 mg/(g·h). The coefficient of variation of the blank controls was 2.3%, which was less than 30% and met the validity of the test.

Under the present test conditions, there were test mixtures with three exposure concentrations (10 mg/L, 100 mg/L, 1000 mg/L). Triplicates of abiotic control with the concentration of 1000 mg/L were tested in the meantime. After an exposure time of three hours, the test results showed that the 3h-EC₅₀ of the test substance for the total oxygen uptake was higher than 1000 mg/L. The average of oxygen uptake rates from the abiotic control was 0 mg/(L·h), which revealed that there was non-obvious abiotic oxygen consumption during the study period.

The absence of inhibition of oxygen consumption by the test substance in the range-finding test demonstrated that a definitive test was unnecessary.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-10-29 to 2019-10-30

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 106 FL TP
Batch number DEF2105728
CAS No. Not definable
Content (certified) 99.63% (w/w)
Water solubility < 1 g/L (OECD 105)
Appearance Slightly yellow flakes
Stability under Not specified
test conditions
Expiry date 2020-01-16
Recommended storage Room temperature. Store in the tightly closed original container at a cool and well ventilated place.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 500 - 1000 mg/L were grounded in a mortar and were weighed out directly in Erlenmeyer flasks and stirred overnight in 200 mL ultrapure water at 400 rpm.
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 500 - 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 30982 Pattensen, Germany)
- Receipt: 2019-10-29
- 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: 2.85 g/L (corresponding to 1.43 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:

19.7 °C

pH:

pH-value of the activated sludge 7.40
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 a mortar, stirring overnight in 200 mL ultrapure water at 400 rpm

- 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,at the end of the test)
- Composition of test medium:

Test Item Test concentration [mg/L]
500 1000
Test Item [mg] 250 500
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 5 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 test item concentrations of 1 – 10 – 100 – 1000 mg test item/L with one replicate each. The pH was measured in one replicate with 1000 mg test item/L. 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 1 – 10 – 100 – 1000 mg test item/L was 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
[%]
1 - -11 -8 -13
10 - -12 -4 -19
100 - -13 -12 -13
1000 7.32 -5 -12 1


No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed.

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 500 and 1000 mg/L in triplicates (limit test to determine the NOEC). The test item was weighed out directly in Erlenmeyer flasks and stirred overnight in 200 mL ultrapure water.

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 with 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 Pattensen was used for the test procedure. It was washed twice and adjusted with chlorine free tap water to 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 for the oxygen uptake rate was determined with the software ToxRat Professional as specified below.
The NOEC was determined by calculation of statistical significance of the oxygen uptake rate in comparison to the control using statistical standard procedure as normality test (Shapiro-Wilk), variance homogeneitiy test (Levene’s test) and a multiple test (Williams Multiple Sequential t-test). Prior to running the multiple test, a normality test and a variance homogeneity test were performed. The P-value for all tests was 0.01. The -value (acceptable probability of incorrectly concluding that there is a difference) is =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)]	[%]	[%]
500	1	8.05	22.0	0	-4
	2	8.05	23.9	-9
	3	8.05	22.9	-4
1000	1	8.19	22.9	-4
	2	8.18	23.3	-6	-3
	3	8.18	21.9	0

*) determined at start of the measurement

                                             Repl. = Replicate

      

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	8.35	14.3	35	31
	2	8.67	14.9	32
	3	8.20	16.1	27
100	1	7.48	11.8	46	45
	2	8.11	12.0	45
	3	6.81	12.6	43
180	1	8.34	3.9	82	82
	2	8.51	3.8	83
	3	8.38	4.1	81

*) determined at start of the measurement

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	8.12	21.8	15.2
	2	7.19	22.5	15.7
	3	8.25	22.1	15.5
	4	8.16	20.3	14.2
	5	7.92	22.7	15.9
	6	8.37	22.5	15.7
	Mean value		22.0	15.4
	Standard deviation		0.81
	CV [%]		3.68

                                                *) determined at start of the measurement

                                               Repl. = Replicate

                                               CV = Coefficient of variation

                                             

    

 

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item LICOCARE RBW 106 FL TP is ≥ 1000 mg/L.
LICOCARE RBW 106 FL TP caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 500 mg/L and 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 106 FL TP (batch number: DEF2105728) from 2019 -10 -29 to 2019 -10 -30, with the definitive exposure phase on 2019 -10 -30 at the test facility. The test system was activated sludge of the municipal treatment plant of 30982 Pattensen, Germany. The test was carried out under static conditions with the limit test item concentrations of 500 and 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. No inhibition of respiration for the test item replicates was detected for 500 and 1000 mg/L.

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 95.6 mg/L. The validity criteria of the test guideline were fulfilled.

The NOEC of the test item LICOCARE RBW 106 FL TP is ≥ 1000 mg/L.

Description of key information

Study 1

The NOEC of the test item LICOCARE RBW 106 FL TP is ≥ 1000 mg/L.
LICOCARE RBW 106 FL TP caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 500 mg/L and 1000 mg/L.

Study 2

Under the present test conditions, 3h-EC₅₀ of the test substance for the total oxygen uptake was higher than 1000 mg/L.