Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

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:

22 March 2017 - 24 March 2017

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 (Carbon and Ammonium Oxidation))

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 03 November 2015

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was not sufficiently soluble to allow the preparation of a 10 g a.i./L stock solution in water. Therefore, 1-litre test bottles were filled with 200 mL of test item mixtures in Milli- RO water with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 24 hours. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
- Controls: medium with sludge and without test item (6 replicates) and one abiotic control without sludge and with the test item (1 replicate)

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands, receiving predominantly
domestic sewage.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test).
- Pretreatment: The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

16 - 23°C

pH:

start: 7.3-7.4
end: 7.3-8.1

Dissolved oxygen:

60-70% saturation

Nominal and measured concentrations:

Nominal concentrations: 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: all glass open bottles/vessels, fill volume: 500 mL
- Aeration: yes, the aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 3 replicates for the highest concentration (1000 mg/L), 1 replicate for other concentrations
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 3.0 g/L after collection; 1.5 g/L in the final test solutions
- Nutrients provided for bacteria: synthetic sewage feed

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap water purified by reverse osmosis)

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: inhibition of respiration at t=3 h

TEST CONCENTRATIONS
- Range finding study: since inhibition respiration rates in the combined limit/range-finding test were 5%, 3% and 28% at 10, 100 and 1000 mg/L, respectively, no further testing was needed.

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

Details on results:

- The combined limit/range-finding test showed 5%, 3% and an average of 28% inhibition of the respiration rate at 10, 100 and 1000 mg/L, respectively. Therefore, the EC50 was above the highest loading rate tested (1000 mg/L).
- The inhibition of the respiration rate of the sludge recorded at a loading rate of 1000 mg D&C Red 6 per litre was statistically significant. Therefore, a NOEC could not be determined.
- The temperature was outside the range prescribed by the study plan. However, because the validity criteria for the controls and reference item were met, this deviation is considered not to have influenced the outcome or validity of the test.
- The reference substance showed that the used batch of sludge had normal sensitivity.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Concentrations tested (nominal): 1.0, 3.2, 10 and 32 mg/L
- Relevant effect levels: EC50: 7.65 mg a.i./L (95% CI: 3.80-11.59)
- Other: result was within the accepted range of 2 to 25 mg/L for total respiration.

Reported statistics and error estimates:

For the reference item calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using a non-linear regression analysis with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
For the test item no EC50-value could be calculated because the effects were below 50%.

NOEC determinaion:
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α=0.05, one-sided, smaller).
Calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1 Results: respiration rate/inhibition, pH values

Replicate	Loading rate (T) Concentration (R) (mg/L)	pH		Respiration rate		% Inhibition respiration rate (mean value)
		Start	End	(mg O2/L.h)	(mg O2/g.h)¹
C 1	0	7.4	8.0	36.99	24.66
C 2	0	7.4	8.0	32.48	21.65
C 3	0	7.4	7.9	36.83	24.55
C 4	0	7.4	7.9	38.25	25.50
C 5	0	7.4	7.9	34.56	23.04
C 6	0	7.4	8.0	28.34	18.89
C Mean				34.58	23.05 (RC)
SD				3.68	2.45
CV (%)				11	11
R 1	1.0	7.4	8.0	36.14	24.09	-4.53
R 2	3.2	7.4	8.1	23.65	15.77	31.60
R 3	10	7.4	8.1	16.46	10.97	52.39
R 4	32	7.4	8.2	2.78	1.85	91.96
T 1	10	7.3	7.9	32.82	21.88	5.08
T 2	100	7.3	7.9	33.41	22.27	3.37
T 3a	1000	7.4	8.1	26.26	17.51	24.05
T 3b	1000	7.4	8.1	23.54	15.69	31.92
T 3c	1000	7.4	8.1	25.19	16.79	27.14
T3 Mean				25.00	16.66 (RT)*	27.70 (IT)
TA	1000	7.4	7.3	0.28	0.19	99.19

C:          Control; R:          Reference item, 3,5-dichlorophenol; T:          Test item, D&C Red 6; TA:       Abiotic control of D&C Red 6; SD:       Standard deviation; CV:       Coefficient of variation

¹             The amount of suspended solids in the final test mixture was 1.5 g/L.

R_C:         Total respiration in the control

R_T:         Total respiration with D&C Red 6

I_T:           % inhibition of total respiration relative to R_C

* :           Statistically significant compared to controls.

Validity criteria fulfilled:

yes

Remarks:

see 'overall remarks' section for details

Conclusions:

Based on the results of a respiration inhibition test, performed according to OECD 209 and GLP principles, the EC50 for D&C Red 6 was determined to be >1000 mg/L. No NOEC could be determined.

Executive summary:

A respiration inhibition test was performed, according to OECD 209 and GLP principles, to assess whether D&C Red 6 was toxic to wastewater bacteria. The test substance was tested at loading rates of 10, 100 and 1000 mg/L for an exposure duration of 3 hours. The highest loading rate was tested in triplicate, the other concentrations consisted of one replicate. Six control vessels and one abiotic control were included. The test substance showed 5%, 3% and an average of 28% inhibition of the respiration rate at 10, 100 and 1000 mg/L, respectively. The EC50 was therefore determined to be >1000 mg/L. No NOEC could be determined since the inhibition of respiration at 1000 mg/L was statistically significant.

Testing with 3,5-dichlorophenol as reference item showed the test system had normal sensitivity. All validity criteria were met and the study was considered to be valid.

Description of key information

A respiration inhibition test was performed, according to OECD 209 and GLP principles, to assess whether D&C Red 6 was toxic to wastewater bacteria. The test substance was tested at loading rates of 10, 100 and 1000 mg/L for an exposure duration of 3 hours. The highest loading rate was tested in triplicate, the other concentrations consisted of one replicate. Six control vessels and one abiotic control were included. The test substance showed 5%, 3% and an average of 28% inhibition of the respiration rate at 10, 100 and 1000 mg/L, respectively. The EC50 was therefore determined to be >1000 mg/L. No NOEC could be determined since the inhibition of respiration at 1000 mg/L was statistically significant.

Testing with 3,5-dichlorophenol as reference item showed the test system had normal sensitivity. All validity criteria were met and the study was considered to be valid.