p-mentha-1,3-diene

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:

26 November 2019 - 02 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test method according to OECD Guideline 209. GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

GLP compliance:

yes (incl. QA statement)

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 directly (using micro-syringes) introduced into the test vessels.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: City of Warsaw's sewage treatment plant ”Czajka”
- Preparation of inoculum for exposure: A sample of an activated sludge was taken from the exit of the aeration tank of the treatment plant ”Czajka”. The sludge was used as collected but coarse particles were removed by settling for a short period and decanting the upper layer of finer solids. The sludge was decanted for a period (15 minutes) to produce a clear supernatant and pellet of sewage solids. The supernatant liquid was discarded and the sludge was resuspended in chlorine-free tap water, with shaking and aeration, and the wash-water was removed by decantation and discarding again. The washing and decantation process was repeated four times. The dry mass of a known volume (100 ml) of the re-suspended sludge was determined and the sludge concentrated by removing liquor or diluted further in chlorine-free tap water to obtain the required sludge solids concentration of 3 g/L. The activated sludge was continuously aerated (1 L/minute) at the test temperature, and was used on the next day. The sludge was fed daily with the synthetic sewage feed (50 mL synthetic sewage feed/L activated sludge) for two additional days. The sludge was then used for the test.
- Initial biomass concentration: 3g/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2°C

pH:

The pH of the mixtures of test item after the 3 hour incubation was between 7.36 and 8.14.

Dissolved oxygen:

60 – 70% saturation

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.17, 1.0, 10.0, 100.0 and 1000.0 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: Glass beakers with a nominal volume of 1000 mL
- Aeration: The beakers were aerated continuously. Clean, oil-free air. Air flow 0.5 to 1 litre/minute.
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six (total respiration), six (heterotrophic respiration) and two (nitrification)
- No. of vessels per abiotic control (replicates): one
- Sludge concentration (weight of dry solids per volume): 1.5 g/L
- Nutrients provided for bacteria: synthetic sewage feed (50 mL synthetic sewage feed/L activated sludge)
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The deionised water was used and chlorine–free tap water for activated sludge washing.
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the exposure period. The concentration of dissolved oxygen was continuously measured and recorded for a 10 minute period, until the oxygen concentration falls below 2 mg/l.

OTHER TEST CONDITIONS
- Adjustment of pH: Yes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Inhibition of total oxygen uptake: At the end of 3 hour incubations, samples were withdrawn to measure the rate of decrease of the concentration of dissolved oxygen in a completely filled BOD bottle. The measurements were made individually. A sample from the first aeration vessel was transferred to fill a BOD bottle and the concentration of dissolved oxygen was immediately measured with the self-stirring oxygen electrode. From the data collected, the specific respiration rates of the control and test mixtures were calculated; the percentage inhibition was then calculated.

Differentiation between inhibition of heterotrophic respiration and nitrification: The use of the specific nitrification inhibitor N-allylthiourea (ATU) enabled the direct assessment of the inhibitory effects of test item on heterotrophic oxidation, and by subtracting the oxygen uptake rate in the presence of ATU from the total uptake rate (no ATU present), the effects on the rate of nitrification were calculated. Two sets of reaction mixtures were prepared, but additionally, ATU was added to each mixture of one set at a final concentration of 11.6 mg/l, which is known to inhibit nitrification completely in sludge with suspended solids concentrations of up to 3000 mg/l. The oxygen uptake rates was measured after the exposure period; these direct values represent heterotrophic respiration only, and the differences between these and the corresponding total respiration rates represent nitrification. The various degrees of inhibition were then calculated.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric serie with ratio of 10

Reference substance (positive control):

yes

Remarks:

(3,5-dichlorophenol)

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

Remarks on result:

other: As in the measured substance content range of 0.17-1000 mg/L the obtained inhibition of dissolved part of test item is less than 50%, it is assumed that EC50 > 1000 mg/L.

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

452 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

271 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

340 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not applicable.
- Effect concentrations exceeding solubility of substance in test medium: the undissolved test item content, visible for test item content above its water solubility, had no significant effect on the inhibition values.
- Adsorption (e.g. of test material to the walls of the test container): not observed.
- Blank controls oxygen uptake rate: >20 mg/gh in control replicates for test item and reference item.
- Coefficient of variation of oxygen uptake rate in control replicates: <30% in control replicates for test item and reference item.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels:
The calculated values of the end points EC50 for the reference item were found to be:
1. Total respiration inhibition: 4.2 mg/L which lies in the range 2 mg/l to 25 mg/l according to validity criteria.
2. Heterotrophic respiration inhibition: 13.4 mg/L which lies in the range 5 mg/l to 40 mg/l of validity criteria.
3. Nitrification inhibition: 1.29 mg/L which is in the range 0.1 mg/l to 10 mg/l according to validity criteria.

Table 5. Test item total respiration; time-related oxygen concentrations (mg/L)

time, min	0.0	1.0	2.0	3.0	4.0	5.0	6.0	7.0	8.0	9.0	10.0
bottle #
1– FB1	7.51	6.80	6.22	5.63	5.06	4.46	3.87	3.27	2.70	2.11	-
2 – FT1/0.17 mg/L	7.50	6.89	6.32	5.74	5.17	4.61	4.03	3.46	2.90	2.31	1.76
3 – FT2/0.17 mg/L	7.55	6.93	6.37	5.80	5.24	4.68	4.12	3.56	2.95	2.41	1.86
4 – FT3/0.17 mg/L	7.55	6.97	6.42	5.86	5.30	4.74	4.18	3.61	3.05	2.48	1.94
5 – FB2	7.54	6.83	6.17	5.53	4.90	4.24	3.63	2.99	2.57	1.99	-
6 – FT1/1.0 mg/L	7.55	6.98	6.41	5.85	5.28	4.70	4.13	3.56	2.99	2.42	1.86
7 – FT2/1.0 mg/L	7.54	6.91	6.35	5.77	5.20	4.61	4.04	3.47	2.89	2.30	-
8 – FT3/1.0 mg/L	7.56	6.94	6.28	5.75	5.17	4.57	3.99	3.39	2.80	2.20	1.63
9 – FB3	7.56	6.91	6.27	5.64	4.99	4.36	3.72	3.09	2.46	1.83	-
10 – FT1/10.0 mg/L	7.50	6.64	6.03	5.42	4.81	4.17	3.55	2.81	2.46	-	-
11 – FT2/10.0 mg/L	7.50	6.76	6.12	5.48	4.83	4.18	3.52	2.88	2.43	-	-
12 – FT3/10.0 mg/L	7.50	6.83	6.18	5.55	4.90	4.25	3.63	2.99	2.35	-	-
13 – FB4	7.50	6.63	5.96	5.27	4.57	3.90	3.21	2.54	1.86	-	-
14 – FT1/100.0mg/L	7.50	6.87	6.23	5.56	4.92	4.27	3.62	2.97	2.34	-	-
15 – FT2/100.0mg/L	7.50	6.35	5.65	4.98	4.30	3.61	2.96	2.29	-	-	-
16 – FT3/100.0mg/L	7.50	6.60	5.95	5.28	4.59	3.89	3.30	2.63	1.99	-	-
17 – FB5	7.50	6.72	6.02	5.29	4.59	3.89	3.15	2.50	1.80	-	-
18 – FT1/1000 mg/L	7.50	6.31	5.67	5.05	4.42	3.81	3.20	2.58	1.96	-	-
19– FT2/1000 mg/L	7.50	6.84	6.22	5.67	5.11	4.53	3.98	3.41	2.84	2.28	-
20 – FT3/1000 mg/L	7.50	6.22	5.63	5.06	4.45	3.88	3.28	2.70	2.11	-	-
21 – FB6	7.50	6.77	6.10	5.41	4.72	4.04	3.34	2.67	2.00	-	-

Table 6. Test item heterotrophic respiration; time-related oxygen concentrations (mg/L)

Time, min	0.0	1.0	2.0	3.0	4.0	5.0	6.0	7.0	8.0	9.0	10.0
bottle #
1 – FB7	8.00	7.33	6.76	6.21	5.80	5.14	4.69	4.23	3.57	3.17	2.35
2 – FHB1	8.20	8.01	7.79	7.58	7.35	7.13	6.92	6.71	6.49	6.27	6.05
3 – FH1/0.17 mg/L	8.20	7.99	7.72	7.51	7.28	7.08	6.88	6.65	6.46	6.25	6.04
4 – FH2/0.17 mg/L	8.20	7.98	7.77	7.54	7.33	7.11	6.89	6.68	6.46	6.24	6.02
5 – FH3/0.17 mg/L	8.20	7.99	7.76	7.56	7.34	7.15	6.92	6.72	6.48	6.27	6.05
6 – FHB2	8.20	7.96	7.71	7.48	7.24	7.02	6.79	6.55	6.33	6.10	5.85
7 – FH1/1.0 mg/L	8.50	8.29	8.09	7.86	7.66	7.45	7.25	7.04	6.82	6.61	6.40
8 – FH2/1.0 mg/L	8.50	8.29	8.08	7.87	7.65	7.44	7.23	7.03	6.82	6.61	6.38
9 – FH3/1.0 mg/L	8.50	8.29	8.08	7.88	7.67	7.46	7.25	7.05	6.85	6.64	6.40
10 – FHB3	8.30	8.06	7.82	7.57	7.34	7.10	6.86	6.63	6.38	6.14	5.90
11 – FH1/10.0 mg/L	8.50	8.28	8.05	7.82	7.60	7.38	7.15	6.95	6.72	6.49	6.27
12 – FH2/10.0 mg/L	8.50	8.27	8.06	7.84	7.62	7.40	7.18	6.96	6.74	6.50	6.28
13 – FH3/10.0 mg/L	8.50	8.28	8.07	7.85	7.63	7.42	7.20	6.98	6.75	6.52	6.30
14 – FHB4	8.30	8.05	7.82	7.59	7.36	7.08	6.84	6.60	6.34	6.10	5.86
15 – FH1/100.0 mg/L	8.50	8.23	7.99	7.76	7.53	7.31	7.08	6.84	6.66	6.37	6.15
16 – FH2/100.0 mg/L	8.50	8.27	8.02	7.81	7.61	7.38	7.14	6.93	6.71	6.49	6.25
17 – FH3/100.0 mg/L	8.50	8.19	7.97	7.76	7.54	7.32	7.10	6.86	6.68	6.40	6.17
18 – FHB5	8.30	8.04	7.79	7.54	7.29	7.03	6.79	6.54	6.27	6.02	5.78
19– FH1/1000 mg/L	8.50	8.30	8.10	7.88	7.66	7.45	7.24	7.03	6.81	6.60	6.37
20 – FH2/1000 mg/L	8.50	8.20	7.97	7.77	7.54	7.34	7.11	6.90	6.68	6.45	6.23
21 – FH3/1000 mg/L	8.50	8.28	8.07	7.85	7.66	7.43	7.22	7.01	6.80	6.57	6.35
22 – FHB6	8.50	8.24	7.99	7.73	7.49	7.23	6.98	6.73	6.48	6.22	5.95
23 – FB8	8.00	7.34	6.73	6.20	5.78	5.18	4.62	4.13	3.55	2.90	2.23

Table 7. Test item. Calculation of percentage of inhibition

Test item total respiration inhibition
Average oxygen consumption rate from FB1 and FB2 equals 36.60 mg/Lh was taken for the calculations
Bottle #	RT. mg/Lh	IT. %
2 – FT1/0.17 mg/L	34.35	6.15
3– FT2/0.17 mg/L	34.14	6.72
4 – FT3/0.17mg/L	33.66	8.03
Average	35.05 ± 0.35	6.97 ± 0.96
Average oxygen consumption rate from FB2 and FB3 equals 37.70 mg/Lh was taken for the calculations
Bottle #	RT. mg/Lh	IT. %
6 – FT1/1.0 mg/L	34.14	9.44
7 – FT2/1.0 mg/L	34.93	7.35
8– FT3/1.0 mg/L	35.73	5.22
Average	34.93 ± 0.79	7.34 ± 2.11
Average oxygen consumption rate from FB3 and FB4 equals 40.25 mg/Lh was taken for the calculations
Bottle #	RT. mg/Lh	IT. %
10 – FT1/10.0 mg/L	35.83	10.98
11 – FT2/10.0 mg/L	37.11	7.80
12 – FT3/10.0 mg/L	38.63	4.02
Average	37.19 ± 1.40	7.60 ± 3.48
Average oxygen consumption rate from FB4 and FB5 equals 42.52 mg/Lh was taken for the calculations
Bottle #	RT. mg/Lh	IT. %
14 – FT1/100.0mg/L	38.70	8.98
15 – FT2/100.0 mg/L	40.60	4.52
16 – FT3/100.0 mg/L	39.51	7.08
Average	39.60 ± 0.95	6.86 ± 2.24
Average oxygen consumption rate from FB5 and FB6 equals 42.00 mg/Lh was taken for the calculations
Bottle #	RT. mg/Lh	IT. %
18 – FT1/1000 mg/L	37.29	11.21
19– FT2/1000 mg/L	34.20	18.57
20 – FT3/1000 mg/L	35.23	16.12
Average	35.57 ± 1.57	15.30 ± 3.75
Test item heterotrophic respiration inhibition
Average oxygen consumption rate from FHB1 and FHB2 equals 13.50 mg/Lh was taken for the calculations
Bottle #	RH. mg/Lh	IH. %
3 – FH1/0.17 mg/L	12.96	4.00
4 – FH2/0.17 mg/L	13.08	3.11
5 – FH3/0.17 mg/L	12.90	4.44
Average	12.98 ± 0.09	3.85 ± 0.68
Average oxygen consumption rate from FHB2 and FHB3 equals 14.01 mg/Lh was taken for the calculations
Bottle #	RH. mg/Lh	IH. %
7 – FH1/1.0 mg/L	13.20	5.78
8 – FH2/1.0 mg/L	13.02	7.07
9 – FH3/1.0 mg/L	13.20	5.78
Average	13.14 ± 0.10	6.21 ± 0.74
Average oxygen consumption rate from FHB3 and FHB4 equals 14.28 mg/Lh was taken for the calculations
Bottle #	RH. mg/Lh	IH. %
11 – FH1/10.0 mg/L	13.38	6.30
12 – FH2/10.0 mg/L	13.32	4.03
13 – FH3/10.0 mg/L	13.20	4.54
Average	13.30 ± 0.09	4.96 ± 1.19
Average oxygen consumption rate from FHB4 and FHB5 equals 14.88 mg/Lh was taken for the calculations
Bottle #	RH. mg/Lh	IH. %
15 – FH1/100.0 mg/L	14.10	5.24
16 – FH2/100.0 mg/L	13.50	9.27
17 – FH3/100.0 mg/L	13.98	6.05
Average	13.86 ± 0.32	6.85 ± 2.13
Average oxygen consumption rate from FHB5 and FHB6 equals 15.21 mg/Lh was taken for the calculations
Bottle #	RT. mg/Lh	IH. %
19– FH1/1000 mg/L	12.78	15.98
20 – FH2/1000 mg/L	13.62	10.45
21 – FH3/1000 mg/L	12.90	15.19
Average	13.10 ± 0.45	13.87 ± 3.00

Table 8. Percentage of test item nitrification inhibition

Test item concentration. mg/L of test item	IN. %
0.17	8.79
1.0	8.02
10.0	8.01
100.0	6.87
1000	16.12

FB: blank control total respiration test substance

FT: total respiration test substance

FH: heterotrophic respiration test substance

FHB: blank control heterotrophic respiration test substance

RT: total respiration

RH: Heterotrophic respiration

IT: percentage inhibition of total oxygen consumption

IH: percentage inhibition of heterotrophic oxygen uptake

An average value of oxygen uptake due to sludge nitrification was 19.53 mg/Lh.

The following end points ECX (mg/L) were determined for total, heterotrophic and nitrification inhibition. The EC50 was expected to be greater than 1000 mg/L.

Inhibition	ECx. mg/L
	EC5	EC10	EC15
Total respiration	-	452	-
Heterotrophic respiration	3.4	271	-
Nitrification	-	340	899

Validity criteria fulfilled:

yes

Remarks:

(results for the reference substance are within the acceptable range; blank controls oxygen uptake rate was less than 20 mg O2/gram of activated sludge in an hour; coefficient of variation of oxygen uptake rate in control replicates is less than 30%)

Conclusions:

The EC50 was determined to be greater than 1000 mg/L as the inhibition of the disolved part of test item was less than 50% for the range of concentrations tested. EC10 was calculated to be 452 mg/L.

Executive summary:

The study of activated sludge respiration inhibition was carried out according with OECD Guideline 209, using activated sludge from a sewage treatment plant treating predominantly domestic sewage. The inhibition of three different oxygen uptakes was determined, total, heterotrophic only and due to nitrification. The microbial inoculum was a preparation of activated sludge adjusted to 1.5 g suspended solids per litre in the test vessels. The respiration rates of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time (incubation) of 3 hours. Five concentrations of the test substance (0.17, 1, 10, 100 and 1000 mg/L) were tested versus blank controls. As positive control substance 3,5-dichlorophenol was used and it was tested in five concentrations (0.01, 0.1, 1.0, 10.0 and 100.0 mg/L). All the validity criteria were found to lie in the ranges allowed. The EC50 was expected to be greater than 1000 mg/L as the inhibition of the disolved part of test item was less than 50% for the range of concentrations tested and the undissolved part, visible for test item content above the substance water solubility, had no significant effect on the inhibition values. The EC10 was calculated to be 452 mg/L.

Description of key information

Key study: Test method according to OECD 209. GLP study: The EC50 was determined to be greater than 1000 mg/L as the inhibition of the disolved part of test item was less than 50% for the range of concentrations tested. EC10 was calculated to be 452 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

452 mg/L