Reaction mass of calcium dihydroxide and calcium peroxide

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:

11/04/2018 - 18/05/2018

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

test item directly weighed into the test vessels.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period and subsequently, synthetic medium, sludge and Milli - RO water were added resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
- Eluate: Milli-RO water
- Controls: blank control, abiotic control, positive control (reference substance).
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas's -Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Pretreatment: The initial pH of the sludge was 5.7 on the day of testing. The sludge was buffered to a pH of 7.0 using a 70 g/L sodium bicarbonate solution. Before the sludge was used, the respiration rate of the sludge was determined to verify the quality of the sludge. The mean respiration rate was 38 mg O2/g.h and therefore, within the validity criterion.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

17 - 22ºC (slightly outside the range prescribed by the study plan for 1.5h, the deviation is considered to have no effect on the outcome of the test).

pH:

The pH in all test vessels, before addition of sludge, was between 7.6 and 9.5. After the 3 hour exposure period the pH was between 6.6 and 10.3.

Dissolved oxygen:

60-70% saturation (> 5 mg/L at 20ºC)

Nominal and measured concentrations:

- range-finding test: 0(control), 10, 100, 1000 mg/L test item (nominal).
- main test: 0 (control), 10, 32, 100, 320, 1000 mg/L test item (nominal).

Details on test conditions:

TEST SYSTEM
- Test vessel: all glass open bottles
- Type (delete if not applicable): open
- Aeration: yes. The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (> 5 mg/L) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 5
- Sludge concentration (weight of dry solids per volume): 3.0 g/L
- Weight of dry solids per volume of reaction mixture per unit of time: 1.5 g/L
- Nutrients provided for bacteria: 50 mL of synthetic medium per litre of activated sludge.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: RO water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)

OTHER TEST CONDITIONS
- Adjustment of pH: no.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Oxygen consumption was measured over a period of 10 min after 3 h incubation of the test vessels. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls. The medium temperature was recorded continuously in temperature control vessels.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study : yes. The final test was performed based on the result of a preceding combined limit/range-finding test.
- Test concentrations: 10, 100 and 1000 mg/L in the range finding test; 10, 32, 100, 320 and 1000 mg/L in the final test.
- Results used to determine the conditions for the definitive study: The combined limit/range-finding showed no inhibition, 24% and 91% inhibition of the respiration rate at a concentration of 10, 100 and 1000 mg/L, respectively. Therefore, the expected EC50 was between 100 and 1000 mg/L. Also, there was a significant oxygen production from the abiotic processes. The oxygen production was 17% of the mean respiration rate of the control. Therefore it was decided to perform the final tests with an addition of an abiotic control of all concentrations.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (Sigma-Aldrich Chemie GmbH, Steinheim, Germany, Lot no. MKBZ0947V)

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

194 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: (95% CL: 165-229 mg/L)

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: (95% CL: 42-82 mg/L)

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

93 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: (95% CL: 69-114 mg/L)

Details on results:

- No statistically significant inhibition of the respiration rate of the sludge was recorded at 32 mg test item. At higher concentrations the inhibitory effect of the test item on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 21% inhibition at 100 mg/L to 97% at 1000 mg/L.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
- Effect concentrations exceeding solubility of substance in test medium: no.
- Adsorption (e.g. of test material to the walls of the test container): no.
- Blank controls oxygen uptake rate: The mean control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (29 mg oxygen per one gram of activated sludge in the final test).
- Coefficient of variation of oxygen uptake rate in control replicates: The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test (18%).

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (3.2 mg/L in the final test).

Reported statistics and error estimates:

Calculation of the ECx value was based on probit analysis using linear maximum likelihood regression, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item. Calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1. Effect parameters for the inhibition of the respiration rate.

Parameter	Test item concentration (mg/L)
NOEC	32
EC10	63 (42 – 82)
EC20	93 (69 – 114 )
EC50	194 (165 – 229)

(  ) Between brackets the 95% confidence intervals are given.

 

Table 2. Combined limit/range-finding Test – Weighed amounts of test item.

Treatment	Concentration (mg/L)	Weighed amount (mg) of test item
		Replicate A	Replicate B	Replicate C
T1	10	5.06	-	-
T2	100	49.75	-	-
T3	1000	499.92	500.02	500.01
TA (abiotic)	1000	499.96	-	-

- :       Not applicable

 

Table 3. Final Test – Weighed amounts of test item.

Treatment	Concentration (mg/L)	Weighed amount (mg) of test item
		Replicate A	Replicate B	Replicate C	Replicate D	Replicate E
T1	10	4.99	5.00	5.02	5.00	5.03
T2	32	16.02	15.96	15.98	15.99	15.96
T3	100	50.01	49.93	49.96	49.96	49.99
T4	320	160.04	160.02	159.96	159.98	159.98
T5	1000	499.77	499.89	499.98	499.90	499.98
TA1	10	4.96	4.98	4.97	5.00	5.02
TA2	32	15.96	15.96	15.97	16.04	15.97
TA3	100	49.98	50.02	49.96	49.97	49.96
TA4	320	159.98	160.04	159.99	159.98	159.96
TA5	1000	499.99	499.69	499.91	499.93	499.98

 

Table 4. Combined Limit/Range-Finding Test – Experimental Set-Up.

Type of bottle	Test/reference item	Synthetic medium	Made up with Milli-RO water	Sludge
	Total volume mixture 250 mL
Control (C)	no	16 mL	yes	250 mL
Reference item concentrations (R )	yes	16 mL	yes	250 mL
Test item concentrations (T)	yes	16 mL	yes	250 mL
Abiotic control of test item, Highest concentration (TA)	yes	16 mL	yes	No sludge, made up to 500 mL with Milli-RO water

  

Table 5. Final – Experimental Set-Up.

Type of bottle	Test/reference item	Synthetic medium	Made up with Milli-RO water	Sludge
	Total volume mixture 250 mL
Control (C)	no	16 mL	yes	250 mL
Reference item concentrations (R)	yes	16 mL	yes	250 mL
Test item1 concentrations (T)	yes	16 mL	yes	250 mL
Abiotic control, test item1 concentrations (TA)	yes	16 mL	yes	No sludge, made up to 500 mL with Milli-RO water

 

Table 6. Final Test – Overview of the oxygen production of abiotic control.

Treatment	Concentration (mg/L)	Mean respiration rate		% uptake, corresponding with the mean control (mean value)
		(mg O2/L h)	(mg O2/g h)¹
TA1	10	-0.54	-0.36	1.26
TA2	32	-0.71	-0.47	1.66
TA3	100	-1.56	-1.04	3.66
TA4	320	-2.22	-1.48	5.19
TA5	1000	-3.43	-2.29	8.03

¹) The amount of suspended solids in the final test mixture was 1.5 g/L

 

Table 7. Final Test – Overview of the Results, corrected for oxygen production in the abiotic control.

Treatment	Concentration (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mg O2/g h)¹
Control		42.77	28.51
T1	10	46.61	31.07	-8.97
T2	32	44.45	29.63	-3.91
T3	100	33.88	22.59	20.78*
T4	320	12.11	8.07	71.68*
T5	1000	1.16	0.77	97.29*

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control.

 

Table 8. Combined Limit/Range-Finding Test – Respiration Rate/Inhibition, pH Values.

Replicate	Concentration (mg/L)	pH		Respiration rate		% Inhibition respiration rate (mean value)
		Start	End	(mg O2/L.h)	(mg O2/g.h)*
C 1	0	7.4	8.3	42.09	28.06
C 2	0	7.4	8.3	43.20	28.80
C 3	0	7.4	8.2	51.88	34.59
C 4	0	7.4	8.3	47.27	31.51
C 5	0	7.4	8.2	48.43	32.29
C 6	0	7.4	8.2	48.37	32.25
C Mean				46.87	31.25(RC)
SD				3.64	2.43
CV (%)				8	8
R 1	1.0	7.4	8.2	54.56	36.37	-16.40
R 2	3.2	7.4	8.4	31.16	20.77	33.52
R 3	10	7.4	8.4	20.79	13.86	55.65
R 4	32	7.4	8.5	8.23	5.49	82.44
T 1	10	7.8	8.3	48.00	32.00	-2.40
T 2	100	9.0	8.5	35.41	23.61	24.46
T 3a	1000	10.0	9.4	3.30	2.20	92.96
T 3b	1000	10.0	9.5	2.90	1.93	93.81
T 3c	1000	9.5	9.4	6.99	4.66	85.09
T3 Mean				4.40	2.93 (RT)*	90.62 (IT)
TA	1000	9.4	9.9	-8.07#	-5.38	117.22

C: Control; R: Reference item, 3,5-dichlorophenol; T: Test item, Reaction mass of calcium dihydroxide and calcium peroxide, EC 930-930-0; TA: Abiotic control of test item; SD: Standard deviation; CV: Coefficient of variation; RT: Total respiration with test item; RC:        Total respiration in the control; IT: % inhibition of total respiration relative to RC; *The amount of suspended solids in the final test mixture was 1.5 g/L. # Oxygen production, therefore expressed as negative value.

 

Table 9. Final Test –Respiration Rate/Inhibition, pH Values.

Replicate	Concentration (mg/L)	pH		Respiration rate		% Inhibition respiration rate (mean value)
		Start	End	(mg O2/L.h)	(mg O2/g.h)*
C 1	0	7.6	7.4	49.61	33.07
C 2	0	7.6	7.2	33.18	22.12
C 3	0	7.6	6.7	52.85	35.23
C 4	0	7.6	6.6	43.77	29.18
C 5	0	7.6	6.7	34.65	23.10
C 6	0	7.6	6.7	42.57	28.38
C Mean				42.77	28.51 (RC)
SD				7.84	5.23
CV (%)				18	18
R 1	1.0	7.6	7.7	35.53	23.69	16.93
R 2	3.2	7.6	8.0	19.45	12.97	54.53
R 3	10	7.6	7.9	12.40	8.27	71.01
R 4	32	7.6	7.9	5.01	3.34	88.29
T 1a	10	8.0	7.3	55.02	36.68	-28.64
T 1b	10	8.0	7.3	44.06	29.37	-3.01
T 1c	10	8.0	7.3	40.73	27.15	4.77
T 1d	10	8.0	7.2	46.69	31.13	-9.16
T 1e	10	8.0	7.2	43.86	29.24	-2.54
T 1 Mean				46.07	30.71 (RT)	-7.72 (IT)
T 2a	32	8.6	7.5	52.89	35.26	-23.66
T 2b	32	8.6	7.4	45.40	30.27	-6.15
T 2c	32	8.6	7.4	41.49	27.66	3.00
T 2d	32	8.6	7.6	34.14	22.76	20.18
T 2e	32	8.6	7.3	44.76	29.84	-4.65
T 2 Mean				43.74	29.16 (RT)	-2.25 (IT)
T 3a	100	8.8	8.0	43.50	29.00	-1.70
T 3b	100	8.9	8.1	32.28	21.52	24.53
T 3c	100	8.9	8.1	26.71	17.81	37.55
T 3d	100	8.9	8.1	32.09	21.39	24.97
T 3e	100	8.9	8.1	27.02	18.01	36.83
T 3 Mean				32.32	21.55 (RT)	24.44 (IT)
T 4a	320	9.0	9.0	11.77	7.85	72.48
T 4b	320	9.0	9.2	9.30	6.20	78.26
T 4c	320	9.0	9.0	8.99	5.99	78.98
T 4d	320	9.0	9.1	9.53	6.35	77.72
T 4e	320	9.0	9.2	9.87	6.58	76.92
T 4 Mean				9.89	6.59 (RT)	76.87 (IT)
T 5a	1000	9.4	9.9	-2.88	-1.92	106.73
T 5b	1000	9.4	9.8	-1.20	-0.80	102.81
T 5c	1000	9.4	9.9	-1.87	-1.25	104.37
T 5d	1000	9.4	9.8	-2.60	-1.73	106.08
T 5e	1000	9.4	9.9	-2.82	-1.88	106.59
T 5 Mean				-2.27#	-1.52 (RT)#	105.32 (IT)

C: Control; SD: Standard deviation; CV: Coefficient of variation; R: Reference item, 3,5-dichlorophenol; T: Test item, Reaction mass of calcium dihydroxide and calcium peroxide, EC 930-930-0; *:         The amount of suspended solids in the final test mixture was 1.5 g/L; RC: Total respiration in the control; RT: Total respiration with test item; IT: % inhibition of total respiration relative to RC; # : Oxygen production, therefore expressed as negative value.

 

 

Table 10. Final Test – Respiration Rate/Inhibition abiotic control, pH Values.

Replicate	Concentration (mg/L)	pH		Respiration rate#		% Inhibition respiration rate (mean value)
		Start	End	(mg O2/L.h)	(mg O2/g.h)*
TA 1a	10	8.0	7.8	-0.38	-0.25	100.89
TA 1b	10	8.0	7.7	-0.06	-0.04	100.14
TA 1c	10	8.0	7.8	-0.45	-0.30	101.05
TA 1d	10	8.0	7.7	-0.74	-0.49	101.73
TA 1e	10	8.0	7.7	-1.06	-0.71	102.48
TA 1 Mean				-0.54	-0.36 (RTA)	101.26 (ITA)
TA 2a	32	8.6	8.0	-0.62	-0.41	101.45
TA 2b	32	8.6	8.0	-0.41	-0.27	100.96
TA 2c	32	8.6	8.0	-0.86	-0.57	102.01
TA 2d	32	8.6	8.0	-1.06	-0.71	102.48
TA 2e	32	8.6	8.0	-0.60	-0.40	101.40
TA 2 Mean				-0.71	-0.47 (RTA)	101.66 (ITA)
TA 3a	100	8.9	8.6	-2.21	-1.47	105.17
TA 3b	100	8.9	8.5	-0.66	-0.44	101.54
TA 3c	100	8.9	8.6	-1.88	-1.25	104.40
TA 3d	100	8.9	8.8	-1.02	-0.68	102.38
TA 3e	100	8.9	8.5	-2.05	-1.37	104.79
TA 3 Mean				-1.56	-1.04 (RTA)	103.66 (ITA)
TA 4a	320	9.1	9.6	-2.94	-1.96	106.87
TA 4b	320	9.1	9.7	-2.78	-1.85	106.50
TA 4c	320	9.1	9.6	-2.47	-1.65	105.77
TA 4d	320	9.1	9.8	-0.86	-0.57	102.01
TA 4e	320	9.1	9.8	-2.05	-1.37	104.79
TA 4 Mean				-2.22	-1.48 (RTA)	105.19 (ITA)
TA 5a	1000	9.4	10.3	-3.45	-2.30	108.07
TA 5b	1000	9.5	10.0	-4.39	-2.93	110.26
TA 5c	1000	9.5	10.1	-2.80	-1.87	106.55
TA 5d	1000	9.5	10.1	-2.71	-1.81	106.34
TA 5e	1000	9.5	9.9	-3.82	-2.55	108.93
TA.5 Mean				-3.43	-2.29 (RTA)	108.03 (ITA)

TA:     Abiotic control of test item; *: The amount of suspended solids in the final test mixture was 1.5 g/L; RTA: Total respiration of the abiotic control with test item; ITA: % inhibition of total respiration of the abiotic control, relative to RC; #: Oxygen production, therefore expressed as negative value.

Table 11. Final Test – Respiration Rate/Inhibition with correction for the abiotic control.

Replicate	Concentration (mg/L)	Respiration rate		% Inhibition respiration rate (mean value)
		(mg O2/L.h)	(mg O2/g.h) #
T1a	10	55.56	37.04	-29.89
T1b	10	44.60	29.73	-4.27
T1c	10	41.27	27.51	3.52
T1d	10	47.23	31.49	-10.42
T1e	10	44.40	29.60	-3.80
T 1 Mean		46.61	31.07 (RT)	-8.97 (IT)
T2a	32	53.60	35.73	-25.32
T2b	32	46.11	30.74	-7.81
T2c	32	42.20	28.13	1.34
T2d	32	34.85	23.23	18.52
T2e	32	45.47	30.31	-6.31
T 2 Mean		44.45	29.63 (RT)	-3.91 (IT)
T3a	100	45.06	30.04	-5.36
T3b	100	33.84	22.56	20.87
T3c	100	28.27	18.85	33.90
T3d	100	33.65	22.44	21.32
T3e	100	28.58	19.06	33.17
T3 Mean		33.88	22.59 (RT)*	20.78 (IT)*
T4a	320	13.99	9.33	67.29
T4b	320	11.52	7.68	73.07
T4c	320	11.21	7.47	73.79
T4d	320	11.75	7.83	72.53
T4e	320	12.09	8.06	71.73
T4 Mean		12.11	8.07 (RT)*	71.68 (IT)*
T5a	1000	0.55	0.37	98.70
T5b	1000	2.23	1.49	94.78
T5c	1000	1.56	1.04	96.34
T5d	1000	0.83	0.56	98.05
T5e	1000	0.61	0.41	98.56
T5 Mean		1.16	0.77 (RT)*	97.29 (IT)*

R: Reference item, 3,5-dichlorophenol; T: Test item; #: The amount of suspended solids in the final test mixture was 1.5 g/L; RT: Total respiration with test item; IT: % inhibition of total respiration relative to RC; *Statistically significantly different compared to control   

  

Table 12. Test Item - Results of probit analysis

Parameter	EC50	EC20	EC10
Value [mg/L]	194.32	92.97	63.24
lower 95%-cl	164.91	68.75	41.98
upper 95%-cl	228.74	114.28	82.42

 

Validity criteria fulfilled:

yes

Remarks:

The mean control oxygen uptake rate was 29 mg O2 per gram of activated sludge in the final test; the coefficient of variation of oxygen uptake in control replicates was 18%; the EC50 of the reference substance was within the acceptable range.

Conclusions:

The 3h-EC50 of the test item on aerobic waste water (activated sludge) bacteria was 194 mg/L, the NOEC was 32 mg/L.

Executive summary:

A study on the activated sludge respiration inhibition of the test item was carried out according to OECD 209 (GLP study). The final test was performed based on the result of a preceding combined limit/range-finding test. The respiration rates of samples of activated sludge from a sewage treatment plant treating predominantly domestic sewage (1.5 g/L suspended solids) and fed with synthetic sewage was measured in an enclosed cell containing an oxygen electrode after a contact time (incubation) of 3 hours. Five concentrations were tested, ranging from 10 to 1000 mg/L and increasing with a factor 3.2. Five replicates per concentration and six replicates for an untreated control group were tested. An abiotic control was performed for all test groups to correct for the oxygen production of the test item (five replicates per group). Since there was an oxygen production observed in the abiotic control with more than 5% in the highest concentration, the respiration rates of the test item are corrected with the respiration rate of the abiotic control from the corresponding concentration. The study met the acceptability criteria. No statistically significant inhibition of the respiration rate of the sludge was recorded at 32 mg/L test item. At higher concentrations, the inhibitory effect of the test item on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 21% inhibition at 100 mg/L to 97% at 1000 mg/L. The 3h-EC50 of the test item on aerobic waste water (activated sludge) bacteria was 194 mg/L (95% CL: 165 - 229 mg/L), the EC20 was 93 mg/L (69 - 114 mg/L) and the EC10 was 63 mg/L (42 - 82 mg/L). The test itemwas not toxic to waste water (activated sludge) bacteria at a concentration of 32 mg/L (NOEC).

Description of key information

Key study. Method according to OECD 209, GLP study. The test item has a 3h-EC50 = 194 mg/L and an EC10 = 63 mg/L in microorganisms. The NOEC was determined to be 32 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

194 mg/L

EC10 or NOEC for microorganisms:

32 mg/L

Additional information

A study on the activated sludge respiration inhibition of the test item was carried out according to OECD 209 (GLP study). The final test was performed based on the result of a preceding combined limit/range-finding test. The respiration rates of samples of activated sludge from a sewage treatment plant treating predominantly domestic sewage was measured in an enclosed cell containing an oxygen electrode after a contact time (incubation) of 3 hours. Five concentrations were tested, ranging from 10 to 1000 mg/L and increasing with a factor 3.2. Five replicates per concentration and six replicates for an untreated control group were tested. An abiotic control was performed for all test groups to correct for the oxygen production of the test item (five replicates per group). Since the oxygen production observed in the abiotic control was above 5% in the highest concentration, the respiration rates of the test item are corrected with the respiration rate of the abiotic control from the corresponding concentration. The study met the acceptability criteria. The 3h-EC50 of the test item on aerobic waste water (activated sludge) bacteria was 194 mg/L (95% CL: 165 - 229 mg/L), the EC20 was 93 mg/L (69 - 114 mg/L) and the EC10 was 63 mg/L (42 - 82 mg/L). The test item was not toxic to waste water (activated sludge) bacteria at a concentration of 32 mg/L (NOEC).