Naphthenic acids, reaction products with diethylenetriamine

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-04-23 to 2012-06-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP, no deficiencies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Naphthenic acids, reaction products with diethylenetriamine

EC Number:

268-610-1

EC Name:

Naphthenic acids, reaction products with diethylenetriamine

Cas Number:

68131-13-5

Molecular formula:

not applicable

IUPAC Name:

Naphthenic acids, reaction products with diethylenetriamine

Details on test material:

- Name of test material (as cited in study report): Napthenic acids, reaction products with diethylenetriamine
- Physical state: Liquid, brown viscous
- Analytical purity: 100 % (UVCB)
- Purity test date: 2011-05-26
- Lot/batch No.: ESD0011831
- Expiration date of the lot/batch: 04/2013
- Stability under test conditions: Not applicable
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Directly weighing of the stock solution, 30 min ultrasound treatment at room temperature
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 10 - 32 - 100 - 320 - 1000 mg/L (factor 3.2) (first study)
3.2 – 10 – 32 mg/L (factor 3.2) (second study)
- Controls: Triplicates at the beginning of the test + triplicates at the end of the test

Test organisms

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: Washed twice with autoclaved tap water and an adjustment to a dry weight concentration of 3.0 g/L +/- 10 % was not necessary. The sludge was used within 24 h after sampling
- Dry sludge concentration: 3.14 g/L (first study); 2.73 g/L (second study)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Hardness:

Not determined

Test temperature:

21.7 °C (first study) and 21.4 °C (second study)

pH:

pH-value of the activated sludge 7.91 first 7.26 second
pH-value of the synthetic waste water 7.56 first 7.44 second

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Composition of test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL demineralised water


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flatbed recorder.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None, limit test concentration
- Range finding study
Inhibition of Respiration (Preliminary Test 1,2 and 3)

Nominal Test Item Concentration
[mg/L] Mean Inhibition
[%]
1st PT 2nd PT 3rd PT
10 10 - -6
100 7 - 54
1000 13 671)
862) 88

Inhibition of Respiration (Preliminary Test 4)

Nominal Test Item Concentration
[mg/L] Mean Inhibition[%]
4th PT
Stirring Stock solution A Stock solution B
500 73 90* 90*

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

117 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CI 103-141 mg/L

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

54.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CI 40-70 mg/L

Effect concentrations 3

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: n.a.

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 94.0 mg/L

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test were 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

The EC-values of the test item were calculated by sigmoidal dose-response regression and the EC50-value of the reference item by linear regression using software GraphPad Prism, GRAPHPAD SOFTWARE, INC. Calculation of the confidence intervals for the EC50-values was carried out using standard procedures.

Any other information on results incl. tables

From the oxygen depletion the oxygen uptake rates were calculated in mg O₂/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O₂/g×h.

The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison with the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration of the Control and Reference Item Concentrations(first study)

Reference Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	36.8	23.4	–	–
	2	36.8	23.4
	3	34.8	22.2
	4	28.4	18.1
	5	29.2	18.6
	6	29.2	18.6
58	1	22.0		32	34
	2	21.6		34
	3	21.2		35
100	1	14.4		56	55
	2	14.4		56
	3	14.8		54
180	1	6.4		80	80
	2	6.4		80
	3	6.8		79

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration of
 the Control and Reference Item Concentrations (second study)

Reference Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	24.4	17.8	–	–
	2	23.6	17.2
	3	24.0	17.5
	4	20.8	15.2
	5	22.0	16.1
	6	20.0	14.6
58	1	16.8		25	26
	2	17.6		22
	3	15.6		31
100	1	10.0		56	54
	2	10.8		52
	3	10.4		54
180	1	4.4		80	81
	2	4.0		82
	3	4.0		82

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration of the Test Item Concentrations and solvent controls (first study)

Test item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Inhibition [%]	Mean Inhibition [%]
*10	1	23.6	27	25
	2	22.4	31
	3	23.6	27
	4	26.0	20
	5	26.8	18
32	1	28.8	11	-2
	2	31.6	3
	3	32.8	-1
	4	36.0	-11
	5	35.6	-10
100	1	16.4	50	41
	2	21.6	34
	3	14.4	56
	4	25.2	22
	5	19.2	41
320	1	4.0	88	89
	2	3.6	89
	3	3.2	90
	4	3.6	89
	5	4.0	88
1000	1	1.6	95	95
	2	1.6	95
	3	1.6	95
	4	1.2	96
	5	1.6	95
Solvent Control 0.64 mL	1	38.4	-18	-11
	2	35.6	-10
	3	34.0	-5
Solvent Control 2 mL	1	21.6	34	32
	2	22.4	31
	3	22.0	32

Oxygen Uptake Rates, Specific Respiration Rates and

Respiration of the Test Item Concentrations and solvent controls (second study)

Test item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Inhibition [%]	Mean Inhibition [%]
3.2	1	28.4	-26	-28
	2	28.4	-26
	3	29.2	-30
	4	29.2	-30
	5	28.4	-26
10	1	26.4	-17	-9
	2	24.8	-10
	3	23.6	-5
	4	23.6	-5
	5	24.4	-8
32	1	28.4	-26	-23
	2	27.2	-21
	3	27.2	-21
	4	28.4	-26
	5	27.2	-21
Solvent Control 0.64 mL	1	28.4	-26	-23
	2	27.6	-23
	3	27.2	-21
Solvent Control 2 mL	1	18.8	16	20
	2	17.6	22
	3	17.6	22

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item is 32 mg/L, the EC10 54.1 mg/L and the EC50 117 mg/L for activated sludge of a municipal sewage treatment plant.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Naphthenic acids, reaction products with diethylenetriamine (batch number: ESD0011831) from 2012-04-23 to 2012-06-19, with the experimental phases on 2012-04-24 and 2012-06-19 at Dr.U.Noack-Laboratorien, D-31157 Sarstedt, Germany. Test system was activated sludge of the municipal treatment plant of D-31137 Hildesheim. The test was carried out under static conditions with the nominal test item concentrations 10 – 32 – 100 – 320 – 1000 mg/L (factor: 3.2). A supplementary study with the concentrations 3.2 – 10 – 32 (factor: 3.2) was carried out to support the NOEC assessment. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates ranged from -2 % to 95 % in the first and -28 % to  -9 % in the second study.

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-values for the reference item were 87.1 mg/L and 94.0 mg/L.

EC-Values and NOEC of Naphthenic acids, reaction products with diethylenetriamine

		Effect values		Confidence interval
		[mg/L]		P = 95 [%] [mg/L]
NOEC		32		n.d.
EC10		54.1		40 – 70
EC20		69.2		54 – 83
EC50		117		103 – 141
EC80		218		160 – 308

                                               n.d.= not determinable

The NOEC of the test item is 32 mg/L, the EC10 54.1 mg/L and the EC50 117 mg/L for activated sludge of a municipal sewage treatment plant.