Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 October 2012 to 18th March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully GLP compliant and in accordance with current test guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

Fatty acids, tall-oil, reaction products with bisphenol A, epichlorohydrin, glycidyl tolyl ether and triethylenetetramine

EC Number:

606-078-8

Cas Number:

186321-96-0

IUPAC Name:

Fatty acids, tall-oil, reaction products with bisphenol A, epichlorohydrin, glycidyl tolyl ether and triethylenetetramine

Details on test material:

Name: TOFA_TETA_PPA_BADGE_CGE_Adduct
Chemical name: Fatty acids, tall-oil, reaction products with bisphenol A, epichlorohydrin, glycidyl tolyl ether and triethylenetetramine
Synonyms: /Euredur 450 BD
CAS number: 186321-96-0
Purity: 100% (UVCB)
Class of compound: Polyaminoamide adduct
Batch number: WA 520
Molecular weight: Mn = 504; Mw = 755; PDI = 1.50 (by GPC)
Expiry Date: end of February 2014
Storage conditions: Tightly sealed container at Room Temperature (15-30ºC)
Hazardous properties: All precautions required in the handling, storage and disposal of the test substance normally taken for a new chemical undergoing safety evaluation will be outlined by the Sponsor.
Disposal: Unused test substance will be returned to the Sponsor or a recipient designated by the Sponsor. Smithers Viscient will document the receipt, distribution and use of the test substance.

Sampling and analysis

Details on sampling:

At the end of the three hour incubation period, a portion of each test preparation was transferred to an appropriate flask. A dissolved oxygen (DO) electrode was sealed in the neck of the flask, ensuring air was completely excluded. The flask contents (total volume 250 mL) were stirred at a constant rate during DO measurements. Oxygen consumption was measured over a period of up to 10 minutes.

Test solutions

Details on test solutions:

Range-Finder Test
Vessels were prepared at 1, 10, 100 and 1000 mg/L nominal concentrations by direct addition of the test substance to each vessel.
Two vessels were treated at all nominal test substance concentrations, with the exception of the 1000 mg/L test level which had three test vessels treated. Four blank controls were also prepared.

Definitive Test
Vessels were prepared at 9.5, 30.5, 97.7, 312.5 and 1000 mg/L nominal concentrations of the test substance to the test vessels. Five vessels were treated at all nominal test substance concentrations. Six blank controls were also prepared.

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

The activated sludge inoculum was collected from the sludge return line at Burley Menston sewage treatment works (West Yorkshire, UK) which has a predominantly domestic catchment. The point of collection was to ensure that the activated sludge sample is relatively free of exogenous material.
The sludge was transported to the test facility in a closed container with an adequate headspace to prevent the sample becoming anoxic. On arrival at the test facility, the sludge was aerated with a compressed air supply delivered through a suitable distribution device.
The suspended solids concentration were determined gravimetrically and adjusted to 3 g/L ± 0.3 g/L. The sludge was maintained at 20 ± 2°C with aeration. During storage the sludge was fed with synthetic sewage concentrate at a rate of 50 mL/L.

Study design

Test type:

not specified

Water media type:

not specified

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not applicable

Test temperature:

The sludge was maintained at 20 ± 2°C during the range-finder and definitive tests with aeration for the incubation period.

pH:

The pH of the sludge was pH 5.77 and was outside the permitted limits of pH 7.5 ± 0.5 and therefore required adjustment with the addition of 2M NaOH, the final pH of the sludge after adjustment was pH 7.33.

Dissolved oxygen:

Dissolved oxygen was measured throughout the test. See results section.

Salinity:

Not applicable

Nominal and measured concentrations:

Range-Finder Test
Vessels were prepared at 1, 10, 100 and 1000 mg/L nominal concentrations, by direct addition of the test substance to each vessel
Definitive Test
Vessels were prepared at 9.5, 30.5, 97.7, 312.5 and 1000 mg/L nominal concentrations, by direct addition of the test substance to the test vessels.

Details on test conditions:

Each vessel contained a synthetic sewage concentrate (in accordance with OECD guideline no. 209), dechlorinated tap water and the test or reference substance as appropriate. Nitrification inhibitor (ATU) was added to appropriate vessels.
The reference substance and nitrification inhibitor (ATU) were added to the test system in aqueous solution where appropriate.
At appropriate intervals, test samples were inoculated and aerated immediately. After addition of the test substance, inoculum and additional water, nominal suspended solids concentration was 1500 mg/L. Each culture was aerated for ca. three hours by bubbling air through the test system. The rate of aeration was sufficient to keep the test samples adequately mixed.
Upon inspection, no observations were recorded to indicate a lack of dispersal or a deposition in the test system inner surfaces as far as was possible given the turbid nature of the test system.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (3,5-DCP)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

157.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Total inhibition

Remarks on result:

other: 108.9 - 312.4 mg/L

Effect concentrations 2

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

9.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Total inhibition

Effect concentrations 3

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

30.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Total inhibition

Details on results:

Variability of results is common in this study design, especially with substances of limited aqueous solubility, and as such statistical analysis and statistical significance is treated with caution (Reference OECD 209 Guideline). For this reason, inhibition of up to 20% is considered to be within normal limits. Considering this, the statistical analysis (p<0.05) observed at all rates in the total respiration endpoint are reported as supporting data. The No Observed Effect Concentration (NOEC) cited here is based on observation and consider a 20% inhibition or enhancement to be within normal variation.
Mean inhibition of 15.84% was noted in the 9.5 mg/L treatment group. The inhibition noted was within normal variation (20%). Therefore, the NOEC was considered to be 9.5 mg/L.
A dose-related inhibition was observed and ranged from 21.8% at 30.5 mg/L to 92.93% at 1000 mg/L.
The EC50 values for inhibition of respiration resulting from the presence of the test item were estimated as follows:
-Total respiration 157.6 mg/L (108.9 to 312.4 fiducial limits)
The EC50 values for inhibition of respiration resulting from the presence of 3,5 DCP were estimated as follows:
-Total respiration 3.92 mg/L

This satisfied the validity criterion that the EC50 of the reference substance was between 2 and 25 mg/L for total respiration.

Reported statistics and error estimates:

Not applicable

Any other information on results incl. tables

Table 1 Range-finder Test Respiration Rate and Inhibition Data

Concentration (mg/L)	Total Respiration Rate (mg O2/L/hr)	Heterotrophic Respiration Rate (mg O2/L/hr)	Nitrification Respiration Rate (mg O2/L/hr)	Total % Inhibition	Heterotrophic % Inhibition	% Nitrification Inhibition
1	84.52	-	-	2.17	-	-
10	79.09	-	-	8.45	-	-
100	72.52	50.59	21.93	16.05	19.53	10.96
1000	12.16	-	-	85.92	-	-

- Not Applicable

 

Control Data

	Total Respiration Rate (mg O2/L/hr) (RTB)	Heterotrophic Respiration Rate (mg O2/L/hr) (RHB)	Nitrification Respiration Rate (mg O2/L/hr) (RNB)
Mean Blank Control Respiration rates (mg O2/L/hr)	86.39	62.87	24.63
Blank Control Respiration Rate CoV(%)	7.57	0.91	12.04
Mean Blank Control Specific Respiration rates (mg O2/g/hr)	57.59	-	-

- Not Applicable

 

Reference Substance

Concentration (mg/L)	Total Respiration Rate (mg O2/L/hr) (RT)	Heterotrophic Respiration Rate (mg O2/L/hr) (RH)	Nitrification Respiration Rate (mg O2/L/hr) (RN)	Total % Inhibition	Heterotrophic % Inhibition	% Nitrification Inhibition
0.1	75.56	60.00	15.56	12.54	4.56	36.85
2	65.88	57.46	8.42	23.74	8.80	65.82
40	9.82	10.00	-0.18	88.64	84.09	100.74

 

Table 2 Definitive Test Respiration Rate and Inhibition Data

Concentration (mg/L)	Total Respiration Rate (mg O2/L/hr)	Total % Inhibition
9.5	59.75	15.84*
30.5	55.52	21.80*
97.7	43.16	39.21*
312.5	24.51	65.48*
1000	5.02	92.93*

* Statistically significant p<0.05 (Wilcoxon/Bonferroni Adj test)

Control Data

Concentration (mg/l)	Total Respiration Rate (mg O2/L/hr)
Mean Blank Control Respiration rates (mg O2/L/hr)	71.00
Blank Control Respiration Rate CoV(%)	12.96
Mean Blank Control Specific Respiration rates (mg O2/g/hr)	47.33

 

Reference Substance

Concentration (mg/L)	Total Respiration Rate (mg O2/L/hr) (RT)	Total % Inhibition
0.1	51.43	27.56
2	42.50	40.14
40	5.41	92.39

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The validity criteria applied to this study type were met and therefore the study is considered valid.
The following endpoints are applied to this study data:
NOEC Total respiration < 9.5 mg/L
LOEC Total respiration 30.5 mg/L
EC50 Total respiration 157.6 mg/L

Executive summary:

The objective of this study was to determine the no observable effect concentration (NOEC) and the 50% effect concentration (EC₅₀) based on inhibition of the rate of oxygen consumption of activated sludge exposed to the test substance, where possible. The study was undertaken according to OECD Guideline 209, Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation), Adopted July, 2010.

A range-finding test, which is not fully reported, was undertaken in order to establish suitable dose levels for the definitive test.

The definitive test concentrations were 9.5, 30.5, 97.7, 312.5 and 1000 mg/L and were achieved by direct addition of the test substance to the test vessels.

The activated sludge inoculum was collected from the sludge return line at Burley Menston Sewage Treatment Works (West Yorkshire, U.K.) which has a predominantly domestic catchment. The point of collection was to ensure that the activated sludge sample was relatively free of exogenous material.

For the definitive test the suspended solids concentration for the sludge was 3.872 g/L which was outside the permitted limits of 3 g/L ± 0.3 g/L and was adjusted to nominal range using dechlorinated water. The pH of the sludge was pH 5.77 and was outside the permitted limits of pH 7.5 ± 0.5 and was therefore adjusted to pH 7.33 with 2M NaOH.

The following endpoints are derived from this study for the test substance TOFA_TETA_PPA_BADGE_CGE Adduct:

	Total Respiration mg/L
NOEC	9.5
LOEC	30.5
EC50	157.6 (108.9 to 312.4 fiducial limits)

The validity criteria applied to this study type were met and therefore the study is considered valid.