trans-cyclohexane-1,2-dinitrilotetraacetic acid

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:

January 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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test item was stored in a closed vessel dry at room temperature (20 ± 5 °C).

Vehicle:

no

Remarks:

Due to the poor solubility of the test item, the test item was added directly into the test vessels.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NW-Lachen-Speyerdorf, Im Altenschemel. Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed /L. In the first experiment (pre-test), the dry matter was determined as 2.92 g suspended solids/L, giving a concentration of 1.46 g suspended solids/L in the test. In the second experiment (main test), the dry matter of the inoculum was determined as 2.18 g suspended solids/L, giving a concentration of 1.09 g suspended solids/L in the test.

Test type:

not specified

Water media type:

other: tap water

Total exposure duration:

3 h

Post exposure observation period:

After 3 hours, the content of the first vessel was poured in a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the O2-concentration over a period of max. 5 minutes. The respiration rate was measured over a period of only 5 minutes. In the guideline, a period of 5 to 10 minutes or until the oxygen concentration falls below 2 mg/L is recommended. As the linearity of all regression linear curves (r2 of 0.99 or more) is given, the measurement period can be considered as sufficiently long.

Hardness:

0.88 mmol/L

Test temperature:

18.5 – 20.7 °C (first experiment (pre-test))
19.1 – 21.4 °C (second experiment (main test))

pH:

The pH of the nutrient solutions was 7.0.
The pH of the water used to prepare the solutions was 8.01.

Conductivity:

210 µS/cm

Nominal and measured concentrations:

In the first experiment (pre-test) the test item was tested using 4 concentrations ranging from 1000 to 1 mg/L (1000, 100, 10 and 1 mg/L) nominal concentration.
In the second experiment (main test) the test item was tested using 5 concentrations ranging from 1000 to 10 mg/L (1000, 320, 100, 32 and 10 mg/L) nominal concentration.

Details on test conditions:

In the blank control vessels, 16 mL nutrient solution (synthetic sewage) was mixed with 234 mL water. The positive control vessels and the treatments were prepared by putting the appropriate amount of positive control solution or test item solution into the respective test vessel, adding 16 mL nutrient solution and water to reach a total volume of 250 mL. Then, 250 mL inoculum was added in 5 minutes intervals and the mixtures were aerated.
After 3 hours, the content of the first vessel was poured in a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the O2-concentration over a period of max. 5 minutes . The following vessels were measured likewise in 5 minutes intervals.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (CAS-No. 591-35-5) was used as positive control. A stock solution in deionised water containing 501 mg/L of 3,5-Dichlorophenol was freshly prepared for each experiment.

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: confidence interval 350 - 860 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

190 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: confidence interval 100 - 270 mg/L

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

For evaluation of the results the nominal concentrations were used because the difference between real concentration and nominal concentration can be stated as not significant.
For the calculation of the EC10 and EC50, the percentage inhibition was plotted versus concentration in a Gauß-logarithmic diagram. EC10 and EC50 were determined from the x values of the regression line at y = 10% and y = 50%.
The coefficient of variation of oxygen uptake rate in blank control replicates was not more than 30% at the end of the test.
The blank controls oxygen uptake rate was more than 20 mg oxygen per gram of activated sludge (dry weight of suspended solids) in 1 hour.

Results with reference substance (positive control):

Concentrations of the positive control were calculated using the concentration of the stock solution and the dilution factor.

Reported statistics and error estimates:

For the treatments with the test item concentrations 1000 and 320 mg/L, it was tested whether the differences between treatment and blank control were significant. For this determination, the values of the O2 consumption were used. The difference between treatment 320 mg/L and the blank control was considered as not significant as the calculated t-value laid below the tabulated t-value and O2 consumption in the treatment was higher than in the blank control. Therefore, the concentration 320 mg/L is stated as NOEC.

Content of Test Vessels in the second experiment (main test):

Vessel No.	Content	Concentration (mg/L) nominal	Nutrient Solution (mL)	Tap Water (mL)	Stock Solution added (mL)	Concen­tration of Stock Solution (mg/L)	Inoculum (mL)
1	Blank Control	0	16	234	0	--	250
2	Blank Control	0	16	234	0	--	250
3	Positive Control	5	16	229	5	501	250
4	Positive Control	10	16	224	10	501	250
5	Positive Control	20	16	214	20	501	250
6	Positive Control	40	16	194	40	501	250
7	Blank Control	0	16	234	0	--	250
8	Blank Control	0	16	234	0	--	250
Vessel No.	Content	Concentration (mg/L) nominal	Nutrient Solution (mL)	Tap Water (mL)	Amount added (mg)	Calculated amount (mg)	Inoculum (mL)
9	Test Item	1000	16	234	500.1	1000.2	250
10	Test Item	1000	16	234	499.8	999.6	250
11	Test Item	1000	16	234	500.3	1000.6	250
12	Test Item	1000	16	234	500.6	1001.2	250
13	Test Item	1000	16	234	499.8	999.6	250
14	Test Item	320	16	234	159.6	319.2	250
15	Test Item	320	16	234	160.4	320.8	250
16	Test Item	320	16	234	160.9	321.8	250
17	Test Item	320	16	234	159.8	319.6	250
18	Test Item	320	16	234	159.6	319.2	250
19	Test Item	100	16	234	50.2	100.4	250
20	Test Item	100	16	234	50.1	100.2	250
21	Test Item	100	16	234	50.2	100.4	250
22	Test Item	100	16	234	50.3	100.6	250
23	Test Item	100	16	234	49.7	99.4	250

Vessel No.	Content	Concentration (mg/L) nominal	Nutrient Solution (mL)	Tap Water (mL)	Amount added (mg)	Calculated amount (mg)	Inoculum (mL)
24	Test Item	32	16	234	15.9	31.8	250
25	Test Item	32	16	234	15.9	31.8	250
26	Test Item	32	16	234	16.1	32.2	250
27	Test Item	32	16	234	16.1	32.2	250
28	Test Item	32	16	234	16.1	32.2	250
29	Test Item	10	16	234	4.9	9.8	250
30	Test Item	10	16	234	5.1	10.2	250
31	Test Item	10	16	234	4.9	9.8	250
32	Test Item	10	16	234	5.1	10.2	250
33	Test Item	10	16	234	4.9	9.8	250
34	Blank Control	0	16	234	--	--	250
35	Blank Control	0	16	234	--	--	250

Results of the second experiment (main test):

Vessel No.	Content	Concentration in mg/L nominal	O2consumption in mg/(L*min)	O2consumption in mg/(L*h)	Inhibition in %	pH meeasured at the end of the test
1	Blank Control	0	0.7024	42.142	0	8.1
2	Blank Control	0	0.7713	46.279	0	8.1
3	Positive Control	5	0.4971	29.826	31.7	8.2
4	Positive Control	10	0.3127	18.764	57.0	8.2
5	Positive Control	20	0.1557	9.345	78.6	8.3
6	Positive Control	40	0.0800	4.799	89.0	8.3
7	Blank Control	0	0.6504	39.022	0	8.2
8	Blank Control	0	0.7296	43.773	0	8.1
9	Test Item	1000	0.0278	1.666	96.2	4.6
10	Test Item	1000	0.0583	3.497	92.0	4.5
11	Test Item	1000	0.0550	3.301	92.4	4.5
12	Test Item	1000	0.0232	1.395	96.8	4.5
13	Test Item	1000	0.0294	1.767	96.0	4.5
14	Test Item	320	0.7550	45.299	-3.7	7.7
15	Test Item	320	0.7025	42.148	3.5	7.8
16	Test Item	320	0.7595	45.569	-4.3	7.8
17	Test Item	320	0.7541	45.245	-3.6	7.8
18	Test Item	320	0.7431	44.587	-2.1	7.8
19	Test Item	100	0.7737	46.420	-6.3	8.0
20	Test Item	100	0.7575	45.452	-4.1	8.0
21	Test Item	100	0.6835	41.011	6.1	8.0
22	Test Item	100	0.7034	42.204	3.4	8.1
23	Test Item	100	0.6415	38.488	11.9	8.1
24	Test Item	32	0.7666	45.997	-5.3	8.1
25	Test Item	32	0.7391	44.346	-1.5	8.1
26	Test Item	32	0.7621	45.727	-4.7	8.1
27	Test Item	32	0.7468	44.810	-2.6	8.1
28	Test Item	32	0.7860	47.163	-8.0	8.0
29	Test Item	10	0.7508	45.048	-3.1	8.1
30	Test Item	10	0.7357	44.141	-1.1	8.1
31	Test Item	10	0.7544	45.265	-3.6	8.1
32	Test Item	10	0.7528	45.168	-3.4	8.1
33	Test Item	10	0.7735	46.411	-6.3	8.1
34	Blank Control	0	0.7763	46.578	0	8.1
35	Blank Control	0	0.7378	44.269	0	8.1

Validity criteria fulfilled:

yes

Remarks:

No observations were made which might cause doubts concerning the validity of the study outcome.

Conclusions:

The following results for the test item CDTA HHQ were determined:
3h NOEC = 320 mg/L
3h EC10 = 190 mg/L (confidence interval 100 - 270 mg/L)
3h EC50 = 500 mg/L (confidence interval 350 - 860 mg/L)

Note: According to the guideline, NOEC is determined by comparing the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower.

Executive summary:

Two experiments were performed.

In the first experiment (pre-test) the test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. For the treatment 1000 mg/L, 5 replicates were used; for the other treatments, 1 replicate each.

Duration of the test was 3 hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 2.92 g suspended solids/L, giving a concentration of 1.46 g suspended solids/L in the test.3,5-Dichloro-phenol was used as positive control.

Because significant inhibition was observed, an additional experiment had to be performed.

In the second experiment (main test) the test item was tested using 5 concentrations ranging from 1000 to 10 mg/L nominal concentration. For all treatments 5 replicates were used. The dry matter of the inoculum was determined as 2.18 g suspended solids/L, giving a concentration of 1.09 g suspended solids/L in the test.

All validity criteria were met. For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. The positive control gave an EC50 of 8.8 mg/L (first experiment pre-test) and 8.4 mg/L (second experiment main test) which lie within the recommended range of 2 – 25 mg/L stated in the OECD guideline. The coefficient of variation of oxygen uptake rate in control replicates was below 30% at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O₂per gram activated sludge in 1 hour.

For evaluation of the results the nominal concentrations were used because the difference between real concentration and nominal concentration can be stated as not significant.

Because no inhibition was observed in the 3 lowest concentrated treatments, the 2 lowest concentrated treatments were not used for evaluation to receive a linear regression.

No observations were made which might cause doubts concerning the validity of the study outcome.

The result of the test can be considered valid.

Description of key information

In the activated sludge respiration inhibition test (OECD Guideline 209) performed on the test item CDTA, the following results were determined:

3h NOEC = 320 mg/L

3h EC10 = 190 mg/L (confidence interval 100 - 270 mg/L)

3h EC50 = 500 mg/L (confidence interval 350 - 860 mg/L)

Key value for chemical safety assessment

EC50 for microorganisms:

500 mg/L

EC10 or NOEC for microorganisms:

190 mg/L