Hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]

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:

1 September 2016 to 22 September 2016

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

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

a stock solution of 10000 mg/L was prepared in deionized water and diluted to get the appropriate concentration with 16 mL nutrient solution and water to give 250 mL. Then, 250 mL inoculum was added.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NW-Lachen-Speyerdorf
- Method of cultivation: aerated and fed with 50 mL synthetic sewage feed /L daily
- storage time: 1 day
- Preparation of inoculum for exposure: filtered and washed 3 times
- Pretreatment: none
- Initial biomass concentration:exp 1: 3.20 g ss/L, exp 2: 2.96 g ss/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

exp 1: 21.3 - 22.1 °C
exp 2: 20.2 - 21.2 °C

Outside the ranges as required in the guidance, but in view of the inhibition found for controls, this has had no effect on the study outcome.

pH:

7.4-7.7

Nominal and measured concentrations:

exp 1: 10, 100 and 1000 mg/L
exp. 2: 100 and 320 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker
- Aeration: continuous
- No. of vessels per concentration (replicates): 5 (exp 1 at 1000 mg/L; exp 2 at 100 and 320 mg/L)
- No. of vessels per control (replicates): 2 replicates before and 2 after measuring positive control and test item, respectively
- No. of vessels per vehicle control (replicates): NA
- No. of vessels per abiotic control (replicates): not included
- Sludge concentration (weight of dry solids per volume): 3.20 and 2.96 for exp 1 and 2 respectively
- Weight of dry solids per volume of reaction mixture per unit of time: 1.60 and 1.48 for exp 1 and 2 respectively
- Nutrients provided for bacteria: pre-test: synthetic sewage feed according to OECD 209
TEST WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Particulate matter: NA
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: NA
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen consumption during a 5 min period (linearity assessed)
TEST CONCENTRATIONS: 100, 30 and 1000 mg/L
- Range finding study: yes at 1, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: 6.7-8.3% inhibition of respiration at 1000 mg/L (statistically significant)

Reference substance (positive control):

yes

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

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

validity criteria were fulfilled

Results with reference substance (positive control):

EC50 8.8 and 8.1 mg/L exp 1 and 2 respectively

Oxygen consumption

Concentration in mg/L	0 (exp 2)	100	320	1000 (exp 1)
Replicates	6	5	5	5
O2consumption in mg/(L*h)	43.33	44.36	41.58	52.058
O2consumption in mg/(L*h)	41.61	45.97	42.77	51.767
O2consumption in mg/(L*h)	44.12	46.38	43.93	51.544
O2consumption in mg/(L*h)	44.04	46.03	44.58	52.453
O2consumption in mg/(L*h)	49.12	46.47	43.71	51.824
O2consumption in mg/(L*h)	48.64	--	--	--
Mean	45.14	45.84	43.31	51.93
Standard Deviation	3.03	0.86	1.16	0.35

 

Respiration inhibition

Concentration in mg/L	0	100	320	1000 (exp 1)
Replicates	6	5	5	5
Percentage inhibition	0	1.7	7.9	7.4
Percentage inhibition	0	-1.8	5.2	7.9
Percentage inhibition	0	-2.7	2.7	8.3
Percentage inhibition	0	-2.0	1.3	6.7
Percentage inhibition	0	-2.9	3.2	7.8
Percentage inhibition	0	--	--	--
Mean	0	-1.54	4.06	7.62

Validity criteria fulfilled:

yes

Conclusions:

The EC50 for inhibition of respiration of bacteria is > 1000 mg/L, the NOEC is 320 mg/L

Executive summary:

In a study according to OECD 209, sludge was exposed to the substance during 3 hours at 100, 320 and 1000 mg/L. At 1000 mg/L a slight inhibition of the respiration was observed (max ca 8%), while at both other concentrations no effect was seen. Therefore it is concluded that the NOEC is 320 mg/L and the EC50 is >1000 mg/L.

Description of key information

In a study according to OECD 209, sludge was exposed to the substance during 3 hours at 100, 320 and 1000 mg/L. At 1000 mg/L a slight inhibition of the respiration was observed (max ca 8%), while at both other concentrations no effect was seen. Therefore it is concluded that the NOEC is 320 mg/L and the EC50 is >1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

320 mg/L