Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 October 1991 to 12 December 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with GLP to available methods at the time. Effects are well documented.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: ETAD - Ecological Method No 103

Deviations:

not applicable

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Department of the Environment, UK (1977). Amenability of sewage sludge to anaerobic digestion

Deviations:

not applicable

Principles of method if other than guideline:

A number of different procedures were used for this work with the overall objectives of assessing potential adverse effects on aerobic and anaerobic sewage treatment processes and the likely degree of bioelimination of Substance HI 12339 in anaerobic processes. The following principles were adopted:

ETAD (Ecological and Toxicological Association of the Dyestuffs Manufacturing Industry). Ecological Method No 103 - A screening test for the Assessment of the Possible Inhibitory Effect of a Chemical Substance on Aerobic Waste-Water Bacteria.
(Note: This ETAD method formed the basis of the OECD Guideline 209 -Activated Sludge, Respiration Inhibition Test).

Department of the Environment, UK (1977). Amenability of sewage sludge to anaerobic digestion. HMSO London

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

EC Number:

413-550-5

EC Name:

Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

Cas Number:

142068-96-0

Molecular formula:

Hill formula: C53H28Cl2N14Na8O26S8

IUPAC Name:

octasodium 5-[(4-chloro-6-{[4-({[4-chloro-6-({7-[2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-8-hydroxy-3,6-disulfonatonaphthalen-1-yl}amino)-1,3,5-triazin-2-yl]amino}methyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]-3-[2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate

Details on test material:

- Name of test material (as cited in study report): V723
- Physical state: Dark brown powder
- Lot/batch No.: NBY 405/74
- Storage condition of test material: darkness at ambient temperature

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 28, 56 & 112 mg/l
Details on sampling are detailed below in "Details on Test Conditions".

Test solutions

Vehicle:

no

Details on test solutions:

See below under "Details on Test Conditions"

Test organisms

Test organisms (species):

other: aerobic and anaerobic sludge.

Details on inoculum:

Not specified within the report.

Study design

Test type:

other: aerobic and anaerobic

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

No post exposure observation

Test conditions

Hardness:

No data

Test temperature:

Variable, dependant on method used. See below under "details on test conditions".

pH:

No data

Dissolved oxygen:

No data

Salinity:

Not applicable.

Nominal and measured concentrations:

- Concentrations: 28, 56 & 112 mg/l (nominal)

Details on test conditions:

4.1 Effect on respiration rate of activated sludge (Study V723/E)

The method used was that defined by the Ecological and Toxicological Association of Dyestuff Manufacturers (ETAD) as Ecological Method 103.

In outline the method consists of aerating a sample of activated sludge for 3 hr in the presence of the test substance and a synthetic sewage, and comparing its respiration rate after this period with that of the same activated sludge aerated for 3 hr in the presence of the synthetic sewage alone.

4.2 Effect on the nitrifying ability of activated sludge (Study V723/D)

The method used was that described by the UK Department of the Environment and measures any adverse effects on the nitrification process in activated sludge.

The experimental procedure involves incubating nitrifying sludge under aerobic conditions for a period of 4 hr in an inorganic medium containing ammonium ion. After this period the concentration of oxidised nitrogen formed in the presence of the test substance at a range of concentrations is compared with controls.

4.3 Effect on sludge digestion and determination of colour removal under anaerobic conditions (Study V723/F)

4.3.1 General description of procedure

The method assesses possible inhibition of anaerobic digestion by measuring the volume of gas produced in the presence and absence of the test substance.

The procedure used was a modification of the Department of the Environment UK method which is currently being amended for testing pure compounds and formulations. The general principles of the modified approach have been described by Owen et al.

For this test, samples of raw feed sludge and sludge from a heated digester were collected from a local sewage treatment works and, after sieving to remove gross particles, the dry sludge solids were determined.

Dry solids (% w/w)

Raw sludge 2.03
Digester sludge 1.03

The two sludges were mixed in the ratio of 1 part raw sludge :3 parts digester sludge. The pH of the raw sludge was adjusted to 7.6 with 2N NaOH and the pH of the mixture was found to be 7.1. The dry solids of the mixture was calculated as 1.28% w/w from the results obtained on the two component sludges.

The mixed sludge was then placed in the incubation bottles of batch digestion units with various amounts of V723 according to the experimental design shown in Table 2.

The incubation bottles were swirled to mix the contents and then allowed to stand for approximately 30 min at 35°C before connection to the gas measuring devices. Each day for the 15 days of the experiment the volume of gas collected was recorded and the results are summarised in Table 3. The temperature of the water bath was also recorded daily and was in the range 33 - 34°C.

4.4 Determination of colour removal

On the day on which the digestion inhibition test ended a sample of the supernatants from each of the test units was taken and analysed by the procedure detailed above.

Reference substance (positive control):

yes

Remarks:

See above under "Details on Test Conditions"

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

>= 100 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 2

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Aerobic sewage treatment

(a) Substance HI 12339 at a nominal test level of 100 mg/1 caused a 2% inhibition of the respiration rate of an activated sludge as determined by the ETAD Method 103 . This inhibition is not considered to be significant and it is unlikely that Substance HI 12339 will affect aerobic sewage treatment.

(b) Substance HI 12339 at a test level of 100 mg/1 caused no inhibition of the nitrifying ability of activated sludge when measured by the standard Department of the Environment (UK) Method (Ref 2). It is unlikely that Substance HI 12339 will affect nitrification during sewage treatment.

Anaerobic sewage treatment

A batch anaerobic digestion experiment was carried out at levels of Substance HI 12339 of up to 4.4% w/w dry sludge solids. No significant inhibition of gas production was observed and it is considered unlikely that Substance HI 12339 will interfere with the sludge digestion process.

Colorimetric analysis of the supernatant at the end of the digestion experiment showed a mean colour removal of 98%.

Results with reference substance (positive control):

4.1 Effect on respiration rate of activated sludge (Study V723/E)

The results are summarised in Table 1 and show 2% inhibition of respiration at V723 test concentrations of 112 mg/I, 56 mg/1 and 28 mg/1. This is not considered to be significant.

The IC50 (concentration for 50% inhibition) of 3,5-dichlorophenol determined on the same batch of activated sludge was 8 mg/1 which is within the normal range (5-20 mg/1) suggested in the ETAD method for this substance.

4.2 Effect on the nitrifying ability of activated sludge (Study V723/D)

NO2 + NO3 formed at 4 hr inhibition (N - mg/I) (%)

Control (triplicate) 19.9

V723: 112 mg/1 (triplicate) 21.2 0

Allylthiourea (11.6 mg/1), used as a standard nitrification inhibitor, gave complete inhibition of nitrification in the same study.

Reported statistics and error estimates:

Not reported.

Any other information on results incl. tables

TABLE 1 - STUDY V723/E: EFFECTS ON RESPIRATION RATE OF ACTIVATED SLUDGE

 

Test unit		Mean respiration rate (mgO2/l/hr)	% inhibition
1,2,14,15	Controls	51.5	-
8,9	112 mg/1 V723	50.5	2
10,11	56 mg/1 V723	50.5	2
12,13	28 mg/1 V723	50.5	2

 

 TABLE 2 - STUDY V723/F: EXPERIMENTAL DESIGN - ANAEROBIC SLUDGE DIGESTION

 

Test unit		Sludge volume (ml)	V723 (mg)^1	Total volume (ml)
1,2,21,22	Controls	300	-	400
15,16	V723 (4.9% on dry solids)	300	189	400
17,18	V723 (2.5% on dry solids)	300	95	400
19,20	V723 (0.89% on dry solids)	300	34	400

 

NOTE

1      The V723 was added by adding the appropriate volume of a 2500 mg/1 standard solution of V723 in water.

TABLE 3 - STUDY V723/F: EFFECTS ON ANAEROBIC SLUDGE DIGESTION

 

		Mean Cumulated gas production (ml) at various time intervals (days)
Test Unit		1	3	5	10	15
1,2,21,22	Controls	70	326	421	523	563
15,16	V723 (4.9% on dry solids)	61	297	387	484	523
15,16	Inhibition	13%	9%	8%	8%	7%
17,18	V723 (2.5% on dry solids)	62	310	403	501	540
17,18	Inhibition	11%	5%	4%	4%	4%
19,20	V723 (0.89% on dry solids)	65	318	413	513	552
19,20	Inhibition	7%	3%	2%	2%	2%

TABLE 4 – STUDY V723/F: DECOLOURISATION – ANAEROBIC SLUDGE DIGESTION

 

Test		Initial concn	Measured concn	%
unit		of V723 (mg/1)	of V723 after 15 days	Decolour-
			digestion (mg/1)	isation
15	V723 (4.9% on dry	473	8.2	98
16	solids)	473	8.0	98
17	V723 (2.5% on dry	238	4.4	98
18	solids)	238	4.3	98
19	V723 (0.89% on dry	85	1.2	99
20	solids)	85	2.4	97

 

Mean decolourisation: 98%

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Substance HI 12339 at a nominal test level of 100 mg/1 caused a 2% inhibition of the respiration rate of an activated sludge as determined by the ETAD Method 103 (Ref 1). This inhibition is not considered to be significant and it is unlikely that Substance HI 12339 will affect aerobic sewage treatment

Substance HI 12339 at a test level of 100 mg/1 caused no inhibition of the nitrifying ability of activated sludge when measured by the standard Department of the Environment (UK) Method (Ref 2). It is unlikely that Substance HI 12339 will affect nitrification during sewage treatment.

The results show 2% inhibition of respiration at V723 test concentrations of 112 mg/I, 56 mg/1 and 28 mg/1. This is not considered to be significant.

The IC50 (concentration for 50% inhibition) of 3,5-dichlorophenol determined on the same batch of activated sludge was 8 mg/1 which is within the normal range (5-20 mg/1) suggested in the ETAD method for this substance

Executive summary:

Study conducted in compliance with GLP and to the following test procedures

1        ETAD (Ecological and Toxicological Association of the Dyestuffs Manufacturing Industry). Ecological Method No 103 - A screening test for the Assessment of the Possible Inhibitory Effect of a Chemical Substance on Aerobic Waste-Water Bacteria.

(Note: This ETAD method formed the basis of the OECD Guideline 209 -Activated Sludge, Respiration Inhibition Test).

2        Department of the Environment, UK (1977). Amenability of sewage sludge to anaerobic digestion. HMSO London

No effects were noticed that were due to the test substance. The substance is not considered to inhibit the respiration or nitrification of sewage sludge microorganisms. EC50 > 100mg/l