cis-2-tert-butylcyclohexyl acetate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 301 F (Ready biodegradability: Manometric Respirometry Test)

Principles of method if other than guideline:

In this ready biodegradability study according to OECD guideline 301F, the toxicity of the test compound to the activated sludge microorganisms was assessed (toxicity control).

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

See "details on test conditions"

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switzerland) was used. The sludge was collected in the morning, washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day. The dry weight of suspended solids of the activated sludge was 4.6 g/L (determined by taking two 50 ml samples of the homogenised sludge, evaporating water on a steam bath, drying in an oven at 105-110 ºC for two hours and weighing the residue). To obtain a concentration of 30 mg/L (dry weight) in a 250 ml flask, 1.63 mL of sludge was needed.

The dry weight of suspended solids was 4.6 g/L, to obatin a concentration of 30 mg/L (dry weight) in a 250 mL flask, 1.63 mL sludge was added to the flasks.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

61 d

Hardness:

no details given in study report

Test temperature:

22 °C

pH:

7.50 - 8.17

Dissolved oxygen:

no details given in study report

Salinity:

no details given in study report

Nominal and measured concentrations:

- Nominal: 100 mg/L
- Measured: not determined

Details on test conditions:

As suggested in the OECD guideline for testing of ready biodegradability (OECD 301F), the toxicity of the test substance for the inoculum was checked. In order to do so, respirometer flasks were filled with mineral medium + inoculum + reference substance + test substance, their respirations recorded and compared to the respiration of flasks containing mineral medium + inoculum + reference substance only.

WATER:
The water used during the study was ultrapure water, containing less than 5 ppb total organic carbon (produced by Millipore Direct-Q 3 UV purification system)

MINERAL MEDIUM:
Prepared by mixing 50 mL of solution A (8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4.2H2O and 0.5 g NH4Cl per litre water) and 2000 mL deionised water, adding 5 mL of each of the solutions B (27.5 g CaCl2 per litre water), C (22.5 g MgSO4.7H2O per litre water) and D (0.25 g FeCl3.6H2O and one drop of HCl conc. per litre water) making up to 5 litres with deionised water. The pH was measured and if necessary adjusted to 7.4 ± 0.2 with phosphoric acid or patassium hydroxide.

PREPARATION OF FLASKS:
The respirometers used during this study (SAPROMAT D 12, J.M. Voith GmbH, Heidenheim, Germany) were used as the test apparaturs, test substance samples (25 mg, corresponding to 100 mg/L in a 250-mL flask) were weighed in small aluminium boats and added directly to the test flasks of the respirometers (SAPROMAT), whereas reference substance samples (sodium benzoate) were added as 1.0 mL of a 25 mg/mL solution in mineral medium.
All flasks were filled with 250 mL of mineral medium. Samples of test reference substance, or both, were added. Then a volume of suspended sludge correspoding to 7.5 mg d.w. was added. Except when the test substance had an acid or alkaline character, the pH of each flask was not measured but assumed to be the same as the mineral medium, in order to not remove any floating undissolved test substance from the test medium by dipping a glass electrode in it. Neutral test substances (as well as sodium benzoate) were shown not to affect the pH of the medium by more than 0.1 pH unit. About 2 g of soda lime was placed in an attachment of the stopper, the flasks closed and placed in the water bath of the respirometer. Glass capillaries were introduced between the test flasks and the oxygen generators to limit losses of the volatile test substance. After temperature and pressure equilibration, the oxygen-meters of the instrument were set to zero (time zero of the experiment).

PERFORMANCE OF THE TEST:
Everyday the oxygen consumption of each flask was recorded and correct temperature and stirring checked. At the end of the test period, the pH of each flask was measured again.

Reference substance (positive control):

yes

Remarks:

benzoic acid, sodium salt (purity: min 99.0%)

Key result

Duration:

61 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The curves obtained with the reference substance alone and those obtained with Agrumex + reference substance show no toxic effect of Agrumex to the micro-organisms at the test concentration of 100 mg/L.

O₂uptake of reference substance (mg O₂/L, adjusted to nominal concentrations).

		Days
		5	7	14	21	28	61
O2uptake sludge (inoculum blanc)	B1	7.0	10.0	19.0	23.0	27.0	43.0
	B2	13.0	15.0	19.0	25.0	30.0	47.0
	B (mean)	10.0	12.5	19.0	24.0	28.5	45.0
O2uptake reference subst. + sludge	A1	137.0	147.0	170.0	178.0	185.0	205.0
	A2	150.0	160.0	181.0	188.0	193.0	209.0
O2uptake reference subst.	A1-B	127.0	134.5	151.0	154.0	156.5	160.0
	A2-B	140.0	147.5	162.0	164.0	164.5	164.0
O2uptake reference subst. + test substance + sludge	E1	156.0	171.0	266.1	304.1	322.1	370.1
	E2	156.0	168.0	239.0	287.0	307.0	357.0
	E (mean)	156.0	169.5	252.5	295.5	314.6	363.6
% biodegradation reference subst.	D1	76	81	91	92	94	96
	D2	84	89	97	98	99	98
	D (mean)	80	85	94	95	96	97

Calculations:

B1, B2, C1, C2, A1, A2, E1, E2: experimental O₂uptake values

B = (B1 + B2) / 2

D1 = 100 * (C1 - B) / ThOD * [S]           Dco1 = 100 * (E1 – A1) / ThOD * [S]

D2 = 100 * (C2 - B) / ThOD * [S]           Dco2 = 100 * (E2 – A2) / ThOD * [S]

D = (D1 + D2) / 2                               Dco = (Dco1 + Dco2) / 2

[S]: Initial test substance concentration (mg/L).

 

Validity criteria fulfilled:

yes

Conclusions:

From the toxicity control of the ready biodegradability test it may be concluded that at a concentration of 100 mg/L, the test substance is not toxic to micro-organisms in activated sludge of (predominantly) domestic sewage. According to REACH guidance R.7B this concentration can be used as a NOEC value.

Executive summary:

The toxicity of the test substance to microorganisms was determined as part of a toxicity control in a GLP-compliant ready biodegradability study according to OECD 301F. In this study activated sludge (obtained from a wastewater treatment plant treating predominantly domestic wastewater) was exposed to 100 mg/L sodium benzoate (positive control) with and without addition of 100 mg/L test substance. The oxygen depletion in both solutions was measured and compared. Inhibition of the degradation of sodium benzoate by the addition of the test substance did not occur. The NOEC for microorganism toxicity is 100 mg/L.

Description of key information

The toxicity of the test substance to microorganisms was determined as part of a toxicity control in a GLP-compliant ready biodegradability study according to OECD 301F. In this study activated sludge (obtained from a wastewater treatment plant treating predominantly domestic wastewater) was exposed to 100 mg/L sodium benzoate (positive control) with and without addition of 100 mg/L test substance. The oxygen depletion in both solutions was measured and compared. Inhibition of the degradation of sodium benzoate by the addition of the test substance did not occur. The NOEC for microorganism toxicity is 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L