Graphene

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:

12 MAY 2021 to 27 JULY 2021

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:

OECD Guidelines for the Testing of Chemicals No. 209 adopted 22. Jul. 2010 ”Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)“

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: Due to the poor solubility of the test item, the test item was weighed directly into the test vessels. In the blank control vessels, 16 mL nutrient solution was mixed with 234 mL water. The positive control vessels and the treatments were prepared by putting the appropriate amount of positive control solution respectively test item solution into the respective test vessel, adding 16 mL nutrient solution and water to reach a total volume of 250 mL. Then, 250 mL inoculum was added in 5 minutes intervals and the mixtures were aerated.
- Controls: 3,5-Dichlorophenol (1,3-Dichloro-5-hydroxybenzene, C6H4Cl2O, CAS-No. 591-35-5) was used as positive control. A stock solution in deionised water containing 500 mg/L was freshly prepared for the experiment. A blank control containing nutrient solution and water only was also included.
- Test concentration separation factor: 10

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected:
The sludge was taken from the activation basin of the sewage treatment plant in 67482 Edenkoben, In den Seewiesen, Germany. The chosen plant treats mostly domestic sewage. The date of collection was June the 9th and the study was started on June the 10th 2021.
- Preparation of inoculum for exposure: On the day before the experiment, the inoculum was taken from its source, washed, aerated and the dry matter was determined. Volume was adapted to the desired content of dry matter. The nutrient solution was thawed and the sludge was fed with 50 mL nutrient solution/L sludge. On the day of the experiment, the dry matter of the inoculumn was determined once more. The stock solution of the positive control was prepared.
- Pre-treatment: Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and resuspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed/L.
- Initial biomass concentration: Dry matter of sludge: 3.08 g suspended solids/L; Dry matter in the test: 1.54 g suspended solids/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

0.90 mmol/L

Test temperature:

18.9 - 21.2 °C

pH:

7.3

Conductivity:

233 pS/cm

Nominal and measured concentrations:

nominal: 0, 1, 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (800 - 1000 mL) were used as test beakers and narrow-neck glass bottles with flat bottoms (250 mL) were used as measuring flasks.
- Aeration: purified air, using Pasteur pipettes
- No. of vessels per concentration (replicates): 1 replicate/concentration for the lower test concentrations (nominal 1, 10 and 100 mg/L) and 5 replicates/concentration for the highest test concentration (nominal 1000 mg/L)
- No. of vessels per control (replicates): 2 replicates before and 2 each after measuring positive control and test item, respectively
- No. of vessels per positive control (replicates): 1 replicate/concentration
- Sludge concentration (weight of dry solids per volume): 3.08 g suspended solids/L (dry matter of sludge)
- Weight of dry solids per volume of reaction mixture per unit of time: 1.54 g suspended solids/L (dry matter in the test)
- Nutrients provided for bacteria: nutrient solution, 16 mL/vessel (please refer to "Any other information on materials and methods incl. tables" for the detailed composition)
- Nitrification inhibitor used (delete if not applicable): none

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
After 3 hours, the content of the first vessel was poured in a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the O2-concentration over a period of max. 5 minutes. The following vessels were measured likewise in 5 minutes intervals.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

Reference substance (positive control):

yes

Remarks:

,5-Dichlorophenol (1,3-Dichloro-5-hydroxybenzene, C6H4Cl2O, CAS-No. 591-35-5)

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

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

All validity criteria were met. The oxygen uptake rate of the blank controls was above 20 mg O2 per gram activated sludge in 1 hour.
No observations were made which might cause doubts concerning the validity of the study outcome.
The result of the test can be considered valid.

Results with reference substance (positive control):

For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. The positive control gave an EC50 of 15 mg/L (95 % confidence interval 9.3 - 27 mg/L) which lie within the recommended range of 2 - 25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30% at the end of the test.

Reported statistics and error estimates:

The oxygen consumption rate is calculated from the slope of the linear part of the oxygen consumption curve using linear regression.
The EC50 value was estimated using the software ORIGIN(TM) 9.0. The data were evaluated using linear fit on a probability-logarithmic scale.
For the determination of the NOEC treatments with the test item concentrations 1000 mg/L were tested for statistical differences compared to the blank control. For this determination, the values of the O2 consumption were used. In order to select a suitable test for significance, it was checked whether equality of variance was given. With the t-test, it was checked whether the differences are significant. Significance is given if the calculated t-value is bigger than the limit of significance (t-value taken from the table with degree of freedom: n1 + n2 – 2, level of significance 95 %).

O₂ Consumption, Inhibition

The values of the O₂consumption (which is a measure for the viability of the bacteria) of control, test and positive control vessels and the calculated inhibition are presented in the following table. The measured pH at the end of the test is also stated.

 

O₂ -Consumption, Inhibition - experiment

Vessel No.	Content	Concentration in mg/L (nominal)	O2consumption in mg/(L*min)	O2consumption in mg/(L*h)	Inhibition in %	pH
1	Blank Control	0	0.6264	37.583	0	7.7
2	Blank Control	0	0.6248	37.487	0	7.8
3	Positive Control	5	0.5996	35.976	4.9	7.9
4	Positive Control	10	0.4497	26.984	28.6	8.0
5	Positive Control	20	0.1568	9.408	75.1	8.1
6	Positive Control	40	0.0843	5.058	86.6	8.1
7	Blank Control	0	0.5557	33.342	0	8.1
8	Blank Control	0	0.6340	38.038	0	8.0
9	Test Item	1000	0.5706	34.234	9.5	8.0
10	Test Item	1000	0.6798	40.791	-7.9	8.0
11	Test Item	1000	0.5674	34.041	10.0	8.1
12	Test Item	1000	0.6235	37.413	1.1	8.1
13	Test Item	1000	0.6185	37.112	1.9	8.1
14	Test Item	100	0.6782	40.694	-7.6	8.0
15	Test Item	10	0.6119	36.711	2.9	8.0
16	Test Item	1	0.6662	39.971	-5.7	8.1
17	Blank Control	0	0.7026	42.156	0	8.0
18	Blank Control	0	0.6377	38.265	0	8.0

 

Biological Results

 

In order to select a suitable test for significance, it was checked whether equality of variance was given. As equality of variances is given, the t-test is used for further calculations.

With the t-test, it was checked whether the differences are significant. Significance is given if the calculated t-value is bigger than the limit of significance (t-value taken from the table with degree of freedom: n1 + n2 – 2, level of significance 95 %).

 

Significance

Concentration	1000 mg/L
calculated t-value t - Test	0.65
tabulated t-value t - Test	2.26
Significance given?	No

 

The difference between treatment 1000 mg/L and the blank control can be considered as not significant as the calculated t-value lay below the tabulated t-value Therefore, the concentration 1000 mg/L is stated as NOEC.

As inhibition in the highest tested treatment was below 50 %, the EC50 is stated as being higher than the highest tested treatment.

As inhibition in the highest tested treatment was below 10 %, the EC10 is stated as being higher than the highest tested treatment.

 

Biological Results Test Item

Parameter	Value	95% Confidence Interval
NOEC	≥ 1000 mg/L	--
3h-EC10	> 1000 mg/L	not determinable
3h-EC50	> 1000 mg/L	not determinable

 

Validity

 

EC50 Positive Control

Date of Experiment	3h-EC50	95% Confidence Interval
10. Jun. 2021	15 mg/L	9.3 – 27 mg/L

 

The EC50 lies within the range of 2 - 25 mg/L.

 

Variation in O₂-Consumption of the Blank Controls

Date ofExperiment	Mean	Standard Deviation	Coefficient of Variation
10. Jun. 2021	37.812 mg/(L*h)	2.802 mg/(L*h)	7.4 %

 

The coefficient of variation of oxygen uptake rate in blank control replicates should not be more than 30% at the end of the test.

The value lies below the recommended limit of 30%.

 

Oxygen Uptake Rate

Date ofExperiment	Oxygen Uptake Rate in mg/h per gram activated sludge
10. Jun. 2021	24.55 mg oxygen per gram sludge in 1 hour

 

The blank controls oxygen uptake rate should not be less than 20 mg oxygen per gram of activated sludge (dry weight of suspended solids) in 1 hour. The value meets the required limit.

Validity criteria fulfilled:

yes

Conclusions:

The activated sludge respiration inhibition test was performed according to OECD TG 209 under GLP without deviations which may have an impact on the validity of the study. Thus, the results were obtained via a scientifically reasonable method. Hence, there is no doubt that the obtained results are not reliable.
According to the reported results no evidence can be found that the test substance has a toxic effect to microorganisms present in activated sludge under the conditions used for the test. Hence, the 3h EC50 was determined to be > 1000 mg/L and the 3h NOEC was determined to be at least 1000 mg/L.

Executive summary:

The present study was performed according to OECD TG 209 under GLP in order to evaluate the effect ofthe registered substance on the respiration of activated sludge obtained from a domestic sewage treatment plant. The Activated Sludge Respiration test is a rapid screening method to identify substances which may adversely affect aerobic micro-organisms.

The effects on micro-organisms from activated sludge were determined by measuring their respiration rate under defined conditions in the presence of different concentrations of the test substance. In this case the test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. The respiration rates of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The activated sludge was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 3.08 g suspended solids/L, giving a concentration of 1.54 g suspended solids/L in the test.

3,5-Dichlorophenol was used as positive control to check the sensitivity of each batch of activated sludge. Four concentrations were tested; an EC₅₀ of 15 mg/L (95% confidence interval: 9.3 – 27 mg/L) was determined, which lies within the recommended range of 2 – 25 mg/L, as stated in the OECD guideline.

One valid experiment was performed.

Because no significant inhibition was observed, no additional experiment had to be performed.

Based on the obtained results, the following results were determined:

3h NOEC >/ = 1000 mg/L

3h EC₁₀ > 1000 mg/L

3h EC₅₀ > 1000 mg/L

Description of key information

The toxicity of the registered substance to microorganism was assessed in an activated sludge respiration inhibition test according to OECD TG 209 under GLP: 3h EC₅₀ > 1000 mg/L, 3h NOEC >/= 1000 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The present study was performed according to OECD TG 209 under GLP in order to evaluate the effect ofthe registered substance on the respiration of activated sludge obtained from a domestic sewage treatment plant. The Activated Sludge Respiration test is a rapid screening method to identify substances which may adversely affect aerobic micro-organisms.

The effects on micro-organisms from activated sludge were determined by measuring their respiration rate under defined conditions in the presence of different concentrations of the test substance. In this case the test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. The respiration rates of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The activated sludge was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 3.08 g suspended solids/L, giving a concentration of 1.54 g suspended solids/L in the test.

3,5-Dichlorophenol was used as positive control to check the sensitivity of each batch of activated sludge. Four concentrations were tested; an EC₅₀ of 15 mg/L (95% confidence interval: 9.3 – 27 mg/L) was determined, which lies within the recommended range of 2 – 25 mg/L, as stated in the OECD guideline.

One valid experiment was performed.

Because no significant inhibition was observed, no additional experiment had to be performed

Based on the obtained results, the following results were determined:

3h NOEC >/ = 1000 mg/L

3h EC₁₀ > 1000 mg/L

3h EC₅₀ > 1000 mg/L