Reaction products of n-octanol and acrylic acid, first distillation pitch

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:

From January 4, 2012 to February 2, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to the OECD 209 Guideline with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10, 32 100, 320 and 1000mg/L
- Sampling method: The synthetic sewage feed (16 mL) and an adequate amount of the test substance loading were mixed and made up to 250 mL with Milli-RO water in a 1 litre bottle. The pH was determined. Thereafter 250 mL activated sludge was added. This was the start of the test. The mixture was then aerated during the contact time, using a pipette as an aeration device.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1-Litre test bottles were filled with 200 mL of test substance mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate.
- Eluate: Milli-RO wate
- Controls: Blank Control, Nitrification control and Abiotic control of test substance.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The batch of sludge was used one day after collection; therefore 50 mL of synthetic 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 sludge was coarsely sieved, washed and diluted with ISO-medium. 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 pH was 7.7 on the day of testing.
- Initial biomass concentration: 3.0 g/L of sludge

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

approximately 10 minutes.

Test temperature:

18.0 - 19.9 ºC

pH:

6.9 - 8.0

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass bottles/vessels.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 1 litre
- Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Biomass loading rate: Initial amount of suspended solids 3.0 g/L of sludge, 1.5 g/L in the final text mixture.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, formulated using RO-water (tapwater purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): the oxygen consumption was recorded for a period of approximately 10 minutes

TEST CONCENTRATIONS
- Range finding study: combined limit/range-finding test
- Test concentrations: 10, 32, 100, 320 and 1000 mg/L
- Results used to determine the conditions for the definitive study: There was no oxygen uptake from abiotic processes and the results at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was slightly higher than the total inhibition. N-allylthiourea was not used in the final test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Conc. based on:

test mat.

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

310 mg/L

Conc. based on:

test mat.

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/L

Conc. based on:

test mat.

Results with reference substance (positive control):

- Results with reference substance valid? yes

Reported statistics and error estimates:

No statistically significant test substance related inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 100 mg Reaction products of n-octanol and acrylic acid, first distillation pitch / litre (Bonferroni t-Test: α=0.05 Toxstat).

Table 1. Final test - Summarized overview of the study results.

Flask	Loading rate (mg/L)	Temp (°C)	pH before addition of sludge	pH after 3 h contact time	Oxygen conc. at the start (~ mg O2/L)	Respiration rate (mg O2/L h)	Respiration rate (mg O2/g h)^1	% Inhibition respiration relative to the control (mean value)
Control	0	19.3­19.9	7.5	7.9	7.6-8.8	49	32	-
T 1	10	18.0­19.1	7.4-7.5	7.8-8.0	6.8-8.9	41	28	15*^2
T 2	32	19.5­19.7	7.4	7.7-7.8	7.0-9.0	50	33	-2
T 3	100	18.0­19.5	7.3-7.4	7.8-7.9	6.8-8.3	45	30	6
T 4	320	18.0­18.7	7.2	7.8-7.9	6.9-8.7	43	28	12*
T 5	1000	18.0­19.5	6.9-7.0	7.7-7.8	7.4-8.8	42	28	14*

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

^*Statistically significant (Bonferroni t-Test: a=0.05 Toxstat).

²This response was not test substance loading rate related and therefore considered as not significant for the test.

Table 2. Effect parameters for the inhibition of the respiration rate.

	Reaction products of n-octanol and acrylic acid, first   distillation pitch
Parameter	Loading rate (mg/L)
NOEC	100
EC10	310
EC20,50,80	>1000

Table 3. Combined limit/range-finding Test – Study results.

 

Flask	Conc./ loading rate (mg/L)	Temp (°C)	pH before addition of sludge	pH after 3 h contact time	Oxygen conc. at the start (~ mg O2/L)	Respiration rate (mg O2/L h)	Respiration rate (mg O2/g h)^1	% Inhibition respiration relative to the control (mean value)
C 1	0	20.7	7.6	7.7	8.4	61	41
C 2	0	19.4	7.5	7.5	8.8	49	33
Mean						55	37
SD						8	6
CV (%)						15	15
CN	0	20.8	7.5	8.2	8.6	36	24	35
R 1	5.0	19.4	7.5	8.1	8.4	30	20	45
R 2	12	20.0	7.5	8.0	8.6	16	11	71
R 3	30	20.0	7.5	8.1	8.9	6	4	89
T1	10	20.7	7.5	7.4	8.8	59	39	-7
T2	100	21.8	7.5	7.2	8.6	53	35	4
T3a	1000	20.0	7.4	7.2	8.3	43	29	22
T3b	1000	20.3	7.4	7.1	8.5	43	29	22
T3c	1000	20.1	7.4	7.1	8.6	41	27	25
T3 mean						42	28	23
TA	1000	19.7	6.9	6.8	8.8	1	1	98
TN	1000	19.1	7.4	7.6	6.7	22	15	39^2

C:	Blank Control
CN:	Nitrification control
R:	Reference substance, 3,5-dichlorophenol
T:	Test substance, Reaction products of n-octanol and acrylic acid, first distillation pitch
CV:	Coefficient of variation
ATU	N-allylthiourea
TA:	Abiotic control of test substance.
TN:	Test substance with ATU.
^1	The amount of suspended solids in the final test mixture was 1.5 g/L.
2	Relative to CN

Table 4. Final Test – Study results.

 

Flask	Conc./ loading rate (mg/L)	Temp (°C)	pH before addition of sludge	pH after 3 h contact time	Oxygen conc. at the start (~ mg O2/L)	Respiration rate (mg O2/L h)	Respiration rate (mg O2/g h)^1	% Inhibition respiration relative to the control (mean value)
C 1	0	19.4	7.5	7.9	7.6	42	28
C 2	0	19.9	7.5	7.9	8.1	50	33
C 3	0	19.4	7.5	7.9	7.7	48	32
C 4	0	19.9	7.5	7.9	7.7	52	35
C 5	0	19.6	7.5	7.9	8.8	50	33
C 6	0	19.3	7.5	7.9	8.3	49	33
Mean						49	32
SD						3	2
CV (%)						7	7
R 1	5.0	19.0	7.5	8.1	7.1	23	15	53
R 2	12	19.3	7.5	8.1	7.5	14	9	71
R 3	30	19.2	7.5	8.2	8.1	7	5	86
T 1a	10	19.1	7.5	7.9	8.2	44	29	9 *^2
T 1b	10	18.7	7.5	7.9	7.8	41	27	15 *^2
T 1c	10	19.0	7.4	7.8	6.8	44	29	9 *^2
T 1d	10	18.0	7.4	7.9	8.9	41	27	15 *^2
T 1e	10	18.1	7.4	8.0	8.6	37	25	24 *^2
T 2a	32	19.7	7.4	7.8	8.2	51	34	-5
T 2b	32	19.7	7.4	7.8	7.9	50	33	-3
T 2c	32	19.5	7.4	7.8	7.0	49	33	-1
T 2d	32	19.5	7.4	7.8	8.8	49	33	-1
T 2e	32	19.6	7.4	7.7	9.0	49	33	-1
T 3a	100	18.4	7.4	7.9	8.0	42	28	13
T 3b	100	18.0	7.4	7.9	7.6	43	29	11
T 3c	100	19.2	7.4	7.8	6.8	51	34	-5
T 3d	100	19.5	7.3	7.8	8.3	48	32	1
T 3e	100	18.2	7.4	7.9	8.0	43	29	11
T 4a	320	18.3	7.2	7.9	8.4	41	27	15 *
T 4b	320	18.0	7.2	7.8	8.2	39	26	20 *
T 4c	320	18.3	7.2	7.8	6.9	46	31	5 *
T 4d	320	18.0	7.2	7.9	8.2	40	27	18 *
T 4e	320	18.7	7.2	7.8	8.7	47	31	3 *
T 5a	1000	19.5	7.0	7.7	8.4	45	30	7 *
T 5b	1000	19.4	7.0	7.7	8.4	44	29	9 *
T 5c	1000	18.0	7.0	7.8	7.4	35	23	28 *
T 5d	1000	18.4	7.0	7.8	8.8	41	27	15 *
T 5e	1000	18.7	6.9	7.7	8.4	44	29	9 *

C: Control

R: Reference substance, 3,5-dichlorophenol

T: test substance, Reaction products of n-octanol and acrylic acid, first distillation pitch

CV: Coefficient of variation

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

* Statistically significant (Bonferroni t-Test: a=0.05 Toxstat)

² This response was not test substance loading rate related and therefore considered as not significant for the test.

Validity criteria fulfilled:

yes

Conclusions:

The test substance was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 100 mg/L (NOEC).

Executive summary:

The activated sludge respiration inhibition test with Reaction products of n-octanol and acrylic acid, first distillation pitch was determinated according to the OECD 209 test guideline and with GLP. The final test was performed based on the result of a preceding combined limit/range-finding test. Five loading rates, ranging from 10 to 1000 mg/L, increasing with a factor 3.2 were tested. Five replicates per loading rate and six replicates for an untreated control group were tested. No statistically significant test substance related inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 100 mg Reaction products of n-octanol and acrylic acid, first distillation pitch / litre. At a loading rate of 320 and 1000 mg/L the inhibition of the respiration rate on aerobic waste water (activated sludge) bacteria was 12 and 14%, respectively. The EC₁₀ value was 310 mg/L and the EC_20,50,80 was > 1000 mg/L.

Description of key information

Key study: OECD Guideline 209. GLP study. The test substance was determined to be not toxic to waste water (activated sludge) bacteria at or below a loading rate of 100 mg/L (NOEC).

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

Key study: Experimental result on test item Reaction products of n-octanol and acrylic acid, first distillation pitch:

The influence of the test substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours according to the OECD 209 Guideline (GLP study). The test substance was determined to be not toxic to waste water (activated sludge) bacteria at or below a loading rate of 100 mg/L (NOEC). The EC₁₀ was at a loading rate of 310 mg/L. The EC_20,50,80 were above the highest loading rate of 1000 mg/L.