Reaction mass of octan-2-ylbenzene, octan-3-ylbenzene and octan-4-ylbenzene

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:

07 may 2019 to 10 May 2019

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

Analytical monitoring:

no

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, the test item was added by direct pipetting into the amber
test bottles containing 200 mL Milli-RO water. The test item – Milli-RO water mixtures were
magnetically stirred for a short period (5-9 minutes). Subsequently, 16 mL synthetic medium,
made up to 50 mL with Milli-RO water, and 250 mL sludge were added, resulting in the
required concentrations (see 'Any other information on materials and methods' for details). Optimal contact between the test item and test
organisms was ensured by applying continuous aeration and stirring.
- Controls: Test medium without test item, treated in the same way as the test item solutions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas',
's-Hertogenbosch, The Netherlands.
- 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.
The concentration of the suspended solids was determined (3 g/L, as used for the test). The pH was 7.5 on the day of testing. 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.
- Medium: Adjusted ISO-medium, formulated using tap water purified by reverse osmosis, with the following composition: CaCl2.2H2O: 211.5 mg/L; MgSO4.7H2O: 88.8 mg/L; NaHCO3: 46.7 mg/L; KCl: 4.2 mg/L.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

Between 20 and 21°C

pH:

At start (T=0h): 7.2 - 7.3
At end (t=3h): 7.1 - 8.2

Nominal and measured concentrations:

Nominal concentration:1.0, 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass open bottles/vessels, 500 mL fill volume
- Aeration: Clean, oil-free air. 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): one replicate for the concetration of 1.0, 10 and 100 mg/L. Three replicates of the 1000 mg/L
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 1.5 g/L suspended solids in the final test mixture.
- Nitrification inhibitor used: none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by reverse osmosis.
- Synthetic medium: Synthetic sewage feed, according to OECD 209.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not specified

EFFECT PARAMETERS MEASURED
- Method: fter the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. 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.
- Oxygen reading: determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.
-Temperature:The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Combined limit/range-finding study: 1.0, 10, 100 and 1000 mg/L

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:

inhibition of total respiration

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

Details on results:

- The combined limit/range-finding test showed no inhibition at 10 and 100 mg/l and an average of 9% inhibition of the respiration rate at 1000 mg/L. Therefore, the EC50 was above
the highest concentration tested (1000 mg/L). See Table 1 in 'Any other information on results' for details on %inhibition.
-The inhibition of the respiration rate of the sludge recorded at a concentration of 1000 mg SHR 1396 per litre was not statistically significant (see also Appendix 3). Therefore, the
NOEC was considered to be at the highest test concentration.
-There was no significant oxygen uptake from abiotic processes.

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (3.2 mg/L with a 95% confidence interval:2.0 - 4.4 mg/L )

Reported statistics and error estimates:

ECx
- reference item: a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis.
- Test item: for SHR 1396, no EC50-value could be calculated because the test item proved to be non-toxic (EC50 > 1000 mg/L).
NOEC determination: (Two-sample t-test Procedure, α=0.05, one-sided, smaller).
Calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table3          
Results - 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)¹
C 1	0	7.3	7.9	40.65	27.10
C 2	0	7.3	8.0	34.60	23.07
C 3	0	7.3	8.0	41.84	27.89
C 4	0	7.3	8.0	38.60	25.73
C 5	0	7.3	7.9	40.01	26.67
C 6	0	7.2	7.9	40.92	27.28
C Mean				39.44	26.29 (RC)
SD				2.60	1.74
CV (%)				7	7
R 1	1.0	7.3	8.2	28.19	18.79	28.52
R 2	3.2	7.3	8.2	20.86	13.91	47.11
R 3	10	7.3	8.2	10.63	7.09	73.05
R 4	32	7.3	8.2	3.18	2.12	91.94
T 1	10	7.3	8.0	37.71	25.14	4.38
T 2	100	7.3	7.9	41.42	27.61	-5.03
T 3a	1000	7.3	7.9	33.95	22.63	13.91
T 3b	1000	7.3	7.7	44.70	29.80	-13.35
T 3c	1000	7.2	7.9	29.00	19.33	26.46
T3 Mean				35.88	23.92 (RT)	9.01 (IT)
TA	1000	7.3	7.1	0.12	0.08

C:          Control

R:          Reference item, 3,5-dichlorophenol       

T:          Test item, SHR 1396             

TA:        Abiotic control of SHR 1396 

SD:        Standard deviation 

CV:       Coefficient of variation                          

R_T:          Total respiration with SHR 1396

R_C:          Total respiration in the control

I_T:           % inhibition of total respiration relative to R_C

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

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test SHR 1396 was not toxic to waste water bacteria (activated sludge) at 1000 mg/L (NOEC).
The EC50 was above 1000 mg/L.

Executive summary:

In a 3 -h activated sludge respiration inhibition study conducted according to OECD Guideline 209 and according to GLP Principles, microorganisms in activated sludge were exposed to the test item at nominal concentrations of 1.0, 10, 100 and 1000 mg/L. The combined limit/range-finding test showed no inhibition at 10 and 100 mg/l and an average of 9% inhibition of the respiration rate at 1000 mg/L.

the EC50 was above the highest concentration tested (1000 mg/L). The inhibition of the respiration rate of the sludge recorded at a concentration of 1000 mg SHR 1396 per litre was not statistically significant. Therefore, the NOEC was considered to be at the highest test concentration (1000 mg/L)

The study met all validity criteria and is considered reliable without restriction.

Description of key information

The EC₅₀ is > 1000 mg/L and the NOEC is 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

In a 3 -h activated sludge respiration inhibition study conducted according to OECD Guideline 209 and according to GLP Principles, microorganisms in activated sludge were exposed to the test item at nominal concentrations of 1.0, 10, 100 and 1000 mg/L. The combined limit/range-finding test showed no inhibition at 10 and 100 mg/l and an average of 9% inhibition of the respiration rate at 1000 mg/L.

the EC₅₀ was above the highest concentration tested (1000 mg/L). The inhibition of the respiration rate of the sludge recorded at a concentration of 1000 mg SHR 1396 per litre was not statistically significant. Therefore, the NOEC was considered to be at the highest test concentration (1000 mg/L)

The study met all validity criteria and is considered reliable without restriction.