4,4'-isopropylidenedi-o-cresol

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Remarks:

Stability of test substance was measured

Details on sampling:

The test substance was stored as follows: room temperature, dark, air-tight in a desiccator.
Sampled before and after exposure

Vehicle:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Konan-Chubu Water Reclamation Plant
- Preparation of inoculum for exposure: MLSS was measured and adjusted to 3000 mg/L by adding ultra pure water on the day of collection. In addition, the synthetic sewage feed was added to the sludge (50mL synthetic sewage feed/1L sludge) and was stored until use for the exposure. The pH was checked and adjusted by adding NaOH to pH 7.5 -/= 0.5 on the day of exposure.
- Storage conditions: 20 +/- 2°C
- Mixed liquor suspended solid concentration (MLSS): 3180 mg/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable

Test temperature:

20 +/- 2°C

Nominal and measured concentrations:

1, 5.6, 32, 180, 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL glass beakers
- Aeration: 0.5 to 1L/min
- No. of vessels per concentration (replicates): 3 (main phase)
- No. of vessels per control (replicates): 6 (main phase)
- Nutrients provided for bacteria: Syntheic sewage feed
- Nitrification inhibitor used: N-allylthiourea
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Ultra pure water
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 3 hour respiration inhibition (based on oxygen consumption).
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 5.62
- Range finding study
- Test concentrations: 1,10, 100 and 1000 mg/L (nominal)
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (DCP)

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:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC50

Basis for effect:

inhibition of nitrification rate

Remarks on result:

not determinable

Remarks:

% of inhibition fluctuated largely. Total respiration was greatly attributed to inhibition of hetrotrophic respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Temperature was maintained at 20.3°C

See tables in Any other information on results incl. tables.

Results with reference substance (positive control):

EC50 of DPC = 7.79 mg/L (Total respiration), 9.3 mg/L (Heterotrophic respiration) and 0.137 mg/L (nitrification respiration). Validity criteria met for reference substance.

Reported statistics and error estimates:

NOEC value was determined by an analysis of variance (ANOVA), Williams test. The significant level for test was set a p=0.05

Table 1: pH values

Concentration (mg/L)	Type of respiration	Total respiration		Type of respiration	Heterotrophic respiration
	No.	pH		No.	pH
		1*	2*		1*	2*
Control	FB-1	7.4	7.7	FBH-1	7.4	7.8
	FB-2	-	7.7	FBH-2	-	7.8
	FB-3		7.7	FBH-3		7.8
	FB-4		7.7	FBH-4		7.8
	FB-5		7.7	FBH-5		7.8
	FB-6		7.7	FBH-6		7.8
1.0	FT1-1	7.4	7.7	FTH1-1	7.4	7.9
	FT1-2	-	7.6	FTH1-2	-	7.8
	FT1-3		7.7	FTH1-3		7.8
5.6	FT2-1	7.4	7.7	FTH2-1	7.4	7.7
	FT2-2	-	7.6	FTH2-2	-	7.8
	FT2-3		7.6	FTH2-3		7.8
32	FT3-1	7.4	7.6	FTH3-1	7.4	7.8
	FT3-2	-	7.6	FTH3-2	-	7.7
	FT3-3		7.6	FTH3-3		7.7
180	FT4-1	7.4	7.5	FTH4-1	7.4	7.9
	FT4-2	-	7.5	FTH4-2	-	7.7
	FT4-3		7.6	FTH4-3		7.7
1000	FT5-1	7.4	7.6	FTH5-1	7.4	7.8
	FT5-2	-	7.5	FTH5-2	-	7.8
	FT5-3		7.5	FTH5-3		7.7

1* Before adding activated sludge

2* At the end of exposure period

Table 2: Oxygen consumption rates of blank controls

Type of respiration	No.	Concentration (mg/L)	Time (min)	DO (mg/L)		R (mgO2/L/hr)		Average	SD	CV (%)
								(mgO2/L/hr)
Total respiration	FB-1	-	0.75	Q1	3.96	RTB-1	46.8	45.2	1.1	2.4
			3.25	Q2	2.01
	FB-2		0.75	Q1	3.41	RTB-2	44.9
			2.58	Q2	2.04
	FB-3		0.75	Q1	3.69	RTB-3	45.3
			2.92	Q2	2.05
	FB-4		0.75	Q1	2.96	RTB-4	43.3
			2.08	Q2	2.00
	FB-5		0.75	Q1	3.22	RTB-5	45.2
			2.33	Q2	2.03
	FB-6		0.75	Q1	4.27	RTB-6	45.6
			3.67	Q2	2.05
Heterotrophic respiration	FBH-1		1.00	Q1	4.99	RHB-1	36.6	40.1	-	-
			5.83	Q2	2.04
	FBH-2		1.00	Q1	5.14	RHB-2	37.6
			6.00	Q2	2.01
	FBH-3		1.00	Q1	4.71	RHB-3	40.9
			4.92	Q2	2.04
	FBH-4		1.00	Q1	5.53	RHB-4	40.7
			6.17	Q2	2.02
	FBH-5		1.00	Q1	4.41	RHB-5	41.3
			4.50	Q2	2.00
	FBH-6		1.00	Q1	4.20	RHB-6	43.4
			4.00	Q2	2.03
Nitrification respiration	-					RNB-1	6.7	5.1	-	-
						RNB-2	4.8
						RNB-3	5.2
						RNB-4	3.2
						RNB-5	5.1
						RNB-6	5.5

 

Equation:

R=(Q1-Q2)/Δtx60

RNB=RTB-RHB (Average)

Table 3: Oxygen consumption rates, percent inhibitions of test mixtures (Total respiration)

Type of respiration	No.	Concentration (mg/L)	Time (min)	DO (mg/L)		R (mgO2/L/hr)		Average (mgO2/L/hr)	Inhibition		Average (%)
Total respiration	FT1-1	1.0	0.75	Q1	4.06	RTT1-1	48.5	46.2	ITT1	-7.3	-2.3
			3.25	Q2	2.04
	FT1-2		0.75	Q1	3.63	RTT1-2	45.3			-0.2
			2.83	Q2	2.06
	FT1-3		0.75	Q1	3.28	RTT1-3	44.9			0.7
			2.42	Q2	2.03
	FT2-1	5.6	0.75	Q1	2.02	RTT2-1	45.6	44.5	ITT2	-0.9	1.5
			2.58	Q2	0.63
	FT2-2		0.75	Q1	3.74	RTT2-2	44.3			2.0
			3.08	Q2	2.02
	FT2-3		0.75	Q1	3.17	RTT2-3	43.7			3.3
			2.33	Q2	2.02
	FT3-1	32	0.75	Q1	2.71	RTT3-1	36.4	40.8	ITT3	19.5	9.8
			3.67	Q2	0.94
	FT3-2		0.75	Q1	1.88	RTT3-2	41.3			8.6
			2.42	Q2	0.73
	FT3-3		0.75	Q1	2.91	RTT3-3	44.6			1.3
			1.92	Q2	2.04
	FT4-1	180	0.75	Q1	3.16	RTT4-1	34.8	31.1	ITT4	23.0	31.1
			2.75	Q2	2.00
	FT4-2		0.75	Q1	2.91	RTT4 -2	28.1			37.8
			2.67	Q2	2.01
	FT4-3		0.75	Q1	3.69	RTT4-3	30.5			32.5
			4.67	Q2	2.04
	FT5-1	1000	0.75	Q1	1.97	RTT-5-1	32.2	31.0	ITT5	28.8	31.5
			3.08	Q2	0.72
	FT5-2		0.75	Q1	2.67	RTT5-2	33.8			25.2
			1.92	Q2	2.01
	FT5-3		0.75	Q1	3.77	RTT5-3	26.9			40.5
			4.67	Q2	2.01

 

Equation:

R=(Q1-Q2)/Δtx60

ITT={1-RTT/RTB (Average)} x 100

Table 4: Oxygen consumption rates, percent inhibitions of test mixtures (heterotropic respiration)

Type of respiration	No.	Concentration (mg/L)	Time (min)	DO (mg/L)		R (mgO2/L/hr)		Average (mgO2/L/hr)	Inhibition		Average (%)
Heterotrophic respiration	FTH1-1	1.0	1.00	Q1	5.67	RHT1-1	40.2	39.3	IHT1	-0.2	2.1
			6.42	Q2	2.04
	FTH1-2		1.00	Q1	5.02	RHT1-2	38.0			5.2
			5.75	Q2	2.01
	FTH1-3		1.00	Q1	4.76	RHT1-3	39.6			1.2
			5.17	Q2	2.01
	FTH2-1	5.6	1.00	Q1	4.26	RHT2-1	38.1	38.2	IHT2	5.0	4.8
			4.50	Q2	2.04
	FTH2-2		1.00	Q1	5.76	RHT2-2	37.7			6.0
			6.92	Q2	2.04
	FTH2-3		1.00	Q1	4.51	RHT2-3	38.7			3.5
			4.83	Q2	2.04
	FTH3-1	32	1.00	Q1	2.20	RHT3-1	28.8	30.9	IHT3	28.2	23.0
			3.50	Q2	1.00
	FTH3-2		1.00	Q1	4.84	RHT3-2	31.1			22.4
			6.42	Q2	2.03
	FTH3-3		1.00	Q1	4.65	RHT3-3	32.7			18.5
			5.83	Q2	2.02
	FTH4-1	180	6.13	Q1	6.13	RHT4-1	23.9	26.2	IHT4	40.4	34.7
			2.54	Q2	2.54
	FTH4-2		5.19	Q1	5.19	RHT4-2	26.4			34.2
			2.00	Q2	2.00
	FTH4-3		4.75	Q1	4.75	RHT4-3	28.3			29.4
			2.00	Q2	2.00
	FTH5-1	1000	5.22	Q1	5.22	RHT-5-1	36.0	28.6	IHT5	10.2	28.7
			2.07	Q2	2.07
	FTH5-2		5.51	Q1	55.1	RHT5-2	25.0			37.7
			2.00	Q2	2.00
	FTH5-3		5.42	Q1	5.42	RHT5-3	24.8			38.2
			2.01	Q2	2.01

 

Equation:

R=(Q1-Q2)/Δtx60

ITT={1-RHT/RHB (Average)} x 100

Table 5: Oxygen consumption rates, percent inhibitions of test mixtures (nitrification respiration)

Type of respiration	No.	Concentration (mg/L)	R (mgO2/L/hr)		Average (mgO2/L/hr)	Inhibition		Average (%)
Nitrification respiration	-	1.0	RNT1-1	9.2	6.9	IHT1	-80.4	-35.9
			RNT1-2	6.0			-17.6
			RNT1-3	5.6			-9.8
	-	5.6	RNT2-1	7.4	6.3	IHT2	-45.1	-24.2
			RNT2-2	6.1			-19.6
			RNT2-3	5.5			-7.8
	-	32	RNT3-1	5.5	9.9	IHT3	-7.8	-93.4
			RNT3-2	10.4			-103.9
			RNT3-3	13.7			-168.6
	-	180	RNT4-1	8.6	4.9	IHT4	-68.6	3.3
			RNT4-2	1.9			62.7
			RNT4-3	4.3			15.7
	-	1000	RNT-5-1	3.6	2.4	IHT5	29.4	53.6
			RNT5-2	5.2			-2.0

Equation:

R=(Q1-Q2)/Δtx60

ITT={1-RNT/RNB (Average)} x 100

Validity criteria fulfilled:

yes

Conclusions:

The 3h EC50 value was calculated to be >1000 mg/L for total respiration inhibition. The NOEC was estimated to be 5.6 mg/L for total respiration inhibition.

Description of key information

OECD guideline, GLP compliant study. Activated sludge was exposed to the test substance for 3 hours during the test. % of respiration inhibition (total, heterotrophic, nitrification) was recorded.

EC50 value of >1000 mg/L and NOEC value of 5.6 mg/L were calculated for total respiration inhibition.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

5.6 mg/L