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Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Remarks:

and nitrification rate

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April the 27th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 22, 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OCSPP 850.3300, "Modified Activated Sludge, Respiration Inhibition Test"

Version / remarks:

January 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

In this test a stock solution was not prepared; however at the start of the test defined amounts of the test item (3, 30 and 300 mg test item corresponding to the concentrations of 10, 100 and 1000 mg/l; furthermore 3 x 300 mg test item for abiotic controls) were directly weighed (administered) into each test flask and the subsequent calculations refer to the initial weighed nominal concentration.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: the (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, one day before the test.
- Pre-treatment of slidge: the coarse particles were removed by settling for 10 minutes and the upper layer of finer solids was decanted. The activated sludge used was washed centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and the wash-water was removed by centrifuging again. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined.
- Initial biomass concentration: based on this ratio, calculated amount of wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to 3 g per litre, based on dry weight.
- Method of cultivation: the activated sludge was not used on the day of the collection, but continuously aerated (2 l/minute) at the test temperature for about 24 hours (1 day) and fed with 50 ml synthetic sewage/l activated sludge.
- pH: the pH of the activated sludge inoculum was checked after preparation (pH: 7.43), pH adjustment of the inoculum was considered not necessary.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.0 - 20.8 °C

pH:

Test vessels: 7.26 - 8.69
Controls: 7.93 - 8.73

Dissolved oxygen:

Test vessels: 7.35 - 8.34 mg O2/l
Controls: 6.59 - 8.32 mg O2/l

Nominal and measured concentrations:

10, 100 and 1000 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer glass bottles of approximately 300 ml volume.
- Aeration: forces aeration by compressed air, 0.5 litre per minute.
- No. of vessels per concentration: 3 replicates were examined at the highest tested concentration of 1000 mg/l (as a limit concentration) and additionally two lower concentration levels of 10 and 100 mg/l were examined with 1 replicate each.
- No. of vessels per blank control: 8 replicates, water; synthetic sewage and inoculum, but without addition of the test or reference item.
- No. of vessels per abiotic control: 3 replicates, tested at the highest test item concentration; water, synthetic sewage and the test item in the corresponding concentration, but without inoculum.
- No. of vessels per reference substance: 1 replicate per concentration; 3,5-Dichlorophenol was tested at the nominal concentrations of 2, 7 and 24.5 mg/l.
- No. of vessels per nitrification: 3 parallels (same as the blank controls, however containing 11.6 mg/l N-allylthiourea) were included in the preliminary experiment.
- Sludge concentration: the activated sludge concentration in all test, reference and blank mixtures was nominally 1.5 g/l of suspended solids.
- Nutrients provided for bacteria: 9.6 ml synthetic sewage.

SYNTHETIC SEWAGE
- Composition: the ratio of components referring to 1000 ml
Peptone 16 g
LAB-LEMCO Powder (BEEF extract) 11 g
Urea 3 g
NaCl 0.7 g
CaCl2 x 2H2O 0.4 g
MgSO4 x 7H2O 0.2 g
K2HPO4 2.8 g
Purified, deionized water ad. 1000 ml
- pH: 7.52, measured on the day of preparation.
- pH adjustment: no additional pH adjustment was necessary.
- Storage: the prepared synthetic sewage was stored in the dark, in refrigerator at the temperature range of 5.1 5.7 °C.

TEST CONDITIONS MONITORING
- Parameters measured: the pH and the oxygen concentrations were determined in all test vessels.
- Measurement time: before the inoculum addition, at the start (just after the inoculation) and at the end of the incubation period in all test concentrations, reference item concentrations and controls.

EFFECT PARAMETERS MEASURED
The oxygen concentration was measured with O2 electrode (working based on LDO method) under stirred conditions and was recorded for about 4-10 minutes. The measurement was carried out in completely filled Winkler bottles.

VALIDITY CRITERIA
- For 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.
- The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30 % at the end of definitive test.
- The 3-hour EC50 of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) should be in the range of 2 to 25 mg/l for total respiration, 5 - 40 mg/l for heterotrophic respiration and 0.1 to 10 mg/l for nitrification respiration.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

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

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

Details on results:

The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect (the observed slight 4.92 % at 10 mg/l, 3.18 % at 100 mg/l and in average -0.48 % at 1000 mg/l inhibitions were within the biological variability of the applied test system) of the test item was observed.
The specific respiration rates of the highest dose, 1000 mg/l were compared with the blank control values using 2 Sample t-Test (α=0.05). No statistical significant differences were observed in the comparison with the blank control values. Based on the results of this study the NOEC was determined to be 1000 mg/l.

FOAMING
In the test the occurring foaming was not significant, controlling was not necessary during the incubation.

NITRIFICATION
With the applying of the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 69.00 mg/lh, the heterotrophic respiration (RH) was 67.57 mg/lh, the nitrification respiration (RN): 1.43 mg/lh was calculated.
The obtained 1.43 mg/lh was considered as not significant difference within a biological variability range of the applied test system, that lower than the 5 % of RT (3.45 mg/lh) in blank controls.
It was assumed that the heterotrophic oxygen uptake equals the total uptake.

ABIOTIC CONTROL
In the experiment the abiotic oxygen consumption of the test substance was zero ( 0.30 and -2.46) in two parallels and negligible (1.26 mg/lh) in one parallel; therefore the total oxygen consumption rates were not corrected with the abiotic control values in the subsequent calculations.

VALIDITY OF THE TEST
The specific respiration rate of the blank controls (without the test substance or reference substance) was 46.00 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 6.98 %.
The 3-hour EC50 of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) was 8.85 mg/l within the range of 5 - 25 mg/l, that was required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary).

Results with reference substance (positive control):

In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 12 % at the lowest concentration of 2 mg/l and at the nominal concentrations of 7 and 24.5 mg/l, the oxygen consumption rate was inhibited by 36 % and 84 %, respectively.
The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 8.85 mg/l.

The Q1, Q2 and the applied Δt values in the Test Item and Control Groups; the Oxygen Consumption Rate (R), and % Inhibition of R

Identifi-cation	Concentration (mg/l)	Oxygen concentration (mg O2/l)		Δt (min)	Oxygen Consumption Rate (R) (mg O2/lh)	Average R (mg O2/lh)	Inhibition of R (%)
		Q1	Q2
CBA	0.00	7.1	2.23	4	73.05	69.00	0.00
CBB		7.21	2.07	5	61.68
CBC		7.54	2.38	4.5	68.80
CBD		7.2	2.27	4	73.95
CBE		7.05	2.21	4	72.60
CBF		7.13	2.32	4	72.15
CBG		7.21	2.17	4.5	67.20
CBH		7.18	2.49	4.5	62.53
CNA	11.6 mg ATU/l	7.45	2.45	4.5	66.67	67.57	2.07
CNB		7.08	2.42	4	69.90
CNC		7.52	2.56	4.5	66.13
R1A	2 mg 3,5-DCP/l	7.26	2.31	5	59.40	60.60	12.17
R1B		7.31	2.17	5	61.68
R1C		7.09	2.03	5	60.72
R2A	7 mg 3,5-DCP/l	7.14	2.16	7	42.69	44.17	35.98
R2B		7.17	2.29	7	41.83
R2C		7.32	2.12	6.5	48.00
R3A	24.5 mg 3,5-DCP/l	7.11	5.28	10	10.98	11.08	83.94
R3B		7.25	5.54	10	10.26
R3C		7.29	5.29	10	12.00
T1/A	10 mg test item/l	7.21	2.29	4.5	65.60	65.60	4.92
T2/A	100 mg test item/l	7.21	2.2	4.5	66.80	66.80	3.18
T3/A	1000 mg test item/l	7.01	2.37	4	69.60	69.33	-0.48^#
T3/B		7.35	2.18	4.5	68.93
T3/C		7.19	2.56	4	69.45
CA1	1000 mg test item/l	7.56	7.61	10	-0.30	-0.50	100.72
CA2		7.6	8.01	10	-2.46
CA3		7.6	7.39	10	1.26

Q1: the oxygen concentration at the beginning of the selected section of the linear phase (mg/L);

Q2: the oxygen concentration at the end of the selected section of the linear phase (mg/L);

Δt: the time interval between these two measurements (min.).

3,5-DCP: 3,5-dichlorophenol

ATU: N-allylthiourea

CV: Coefficient of variation

#: The value was taken into consideration as zero.

The Specific Respiration Rate (RS) in the Test Item and Control Groups

Identification	Concentration (mg/l)	Specific Respiration Rate (mg O2/gh)	Average RS(mg O2/gh)
CBA	0.00	48.70	46 CV(%) = 6.98
CBB		41.12
CBC		45.87
CBD		49.30
CBE		48.40
CBF		48.10
CBG		44.80
CBH		41.69
CNA	11.6 mg ATU/l	44.44	45.04 CV(%) = 1.54
CNB		46.60
CNC		44.09
R1A	2 mg 3,5-DCP/l	39.60	40.4 CV(%) = 1.89
R1B		41.12
R1C		40.48
R2A	7 mg 3,5-DCP/l	28.46	29.45 CV(%) = 7.57
R2B		27.89
R2C		32.00
R3A	24.5 mg 3,5-DCP/l	7.32	7.39 CV(%) = 7.89
R3B		6.84
R3C		8.00
T1/A	10 mg Test Item/l	43.73	43.73
T2/A	100 mg Test Item/l	44.53	44.53
T3/A	1000 mg Test Item/l	46.40	46.22^n.s.CV(%) = 0.50
T3/B		45.96
T3/C		46.30
CA1	1000 mg Test Item/l	-0.20	-0.33 CV(%) F32= -373.61^##
		-1.64
		0.84

Validity criteria fulfilled:

yes

Remarks:

for the black control oxygen uptake was > 20 mgO2/g/h and the control coefficient of variation was < 30 %. the EC50 (3h) of reference substance resulted in the suitable ranges for total respiration, heterotrophic respiration and nitrification

Conclusions:

EC50 (3h) > 1000 mg/l (nominal)
NOEC (3h): 1000 mg/l (nominal)

Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions. The respiration rates (total, heterotrophic and nitrification oxygen uptake 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 preliminary test was used to estimate the range of concentrations of the test item needed in a possible definite test for determining the inhibition of oxygen consumption. The test item was investigated in the study at the nominal concentrations of 10; 100 and 1000 mg/l. In parallel with the test item treatments 3,5-Dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/l; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated.

Abiotic controls (investigated in three parallels) were prepared containing the test item in the concentration of 1000 mg/l, synthetics sewage feed, but no inoculum.

The average specific respiration rate of the blank was 46.00 mg O₂/g activated sludge (based on dry weight) in an hour with a coefficient of variation of 6.98 %. The 3-hour EC50 of the reference item 3,5-Dichlorophenol was 8.85 mg/l within the range of 5 to 25 mg/l, that was required for total respiration.

The observed oxygen consumption rates consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls (the average specific respiration rate at 1000 mg/l: 46.22 mg O₂/g), no inhibitory effect of the test item was observed.

Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are greater than 1000 mg/l. The NOEC was determined to be 1000 mg/l.

In conclusion, this preliminary test demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/l, therefore a definite test was considered unnecessary.

Conclusion

EC50 (3h) > 1000 mg/l (nominal)

NOEC (3h): 1000 mg/l (nominal)

Description of key information

Non toxic to microorganisms

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are greater than 1000 mg/l. The NOEC was determined to be 1000 mg/l.

In conclusion, this preliminary test demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/l, therefore a definite test was considered unnecessary.