[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted on 19 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to OECD TG 209 in c ompliance with GLP, without deviations that influence the quality of the results

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

EC Number:

289-348-4

EC Name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

Cas Number:

87788-32-7

Molecular formula:

C10H22B2F6N2

IUPAC Name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

impurity 1

Reference substance name:

{3-[(amino-N)methyl]-3,5,5-trimethylcyclohexanamine}(trifluoro)boron

Molecular formula:

C10H22BF3N2

IUPAC Name:

{3-[(amino-N)methyl]-3,5,5-trimethylcyclohexanamine}(trifluoro)boron

additive 1

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

Test material form:

liquid

Specific details on test material used for the study:

Identification: (μ(5-amino-1,3,3-trimethylcycloihexylamine-N,N')hexafluorodiboron
Physical state/Appearance: clear colorless liquid
Batch: AEF0009100
Purity: unknown
Expiry Date: 29 August 2017
Storage Conditions: room temperature in the dark
Intended use/Application: substance used in industry

Sampling and analysis

Analytical monitoring:

no

Remarks:

As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and is reflected in the GLP compliance statement.

Test solutions

Vehicle:

no

Details on test solutions:

Range-Finding Test
Test Item Preparation
In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dissolved directly in water.
A nominal amount of test item (2500 mg) was dissolved in water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 liter to give a
2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and
1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity.
The pH of the test item stock solutions were measured using a Hach HQ40d Flexi handheld meter (see Table 1) and adjusted to between pH 7.0 to pH 8.0 if necessary.


Reference Item Preparation
A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 17 minutes. The pH of this stock solution was measured to be pH 5.8 and was adjusted to pH 7.2 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 19 October 2016 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

Preparation of Inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (4.5 liters) was fed synthetic sewage (225 mL). The pH of the sample on the day of the test was 7.6 measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper* using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Hardness:

Not reported

Test temperature:

20”C

pH:

7.3 - 7.9

Dissolved oxygen:

5.7 - 8.6 mg O2/L or 64 - 97% of Dissolved Oxygen Saturation Level of 8.9 mg O2/L

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Range-Finding Test
Nominal test concentrations of 10, 100 and 1000 mg/L

Details on test conditions:

Preparation of Test System
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. The test item vessels were prepared as described above above under "Details of Test Solutions". Finally two further control vessels were prepared.

Reference substance (positive control):

yes

Remarks:

Information as provided by the Supplier (Sigma-Aldrich). Identification: 3,5-Dichlorophenol Physical state/Appearance: pale brown crystalline solid Batch: MKBV7274V Purity: 99.6% Expiry Date: 01 September 2017 Storage Conditions: room temperature

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Range-Finding Test
The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 1. Oxygen consumption rates and percentage inhibition values for the control, test and reference item after 3 hours contact time are given in Table 2.
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.
No statistically significant toxic effects were shown at the test concentration of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Validation Criteria
The coefficient of variation of oxygen uptake in the control vessels was 7.8% and the specific respiration rate of the controls was 27.00 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.
The validation criterion for the reference item EC50 value was also satisfied.

Inhibition of Respiration Rate
The following results were derived:
FAT 93588/A TE 3,5-dichlorophenol
ECx (3 Hours) (mg/L) 95% Confidence Limits (mg/L) ECx (3 Hours) (mg/L) 95% Confidence Limits (mg/L)
EC10 100 - 1000 - 1.9 -
EC20 100 - 1000 - 2.7 -
EC50 >1000 7.7 6.1-9.8
EC80 >1000 - 22 -
NOEC 100 - - -

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Results with reference substance (positive control):

See "Details on Results" (above)

Any other information on results incl. tables

Table 1: Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L
Control	R1	6.2	70
	R2	6.0	67
	R3	5.9	66
	R4	6.3	71
Test Item	10	5.7	64
	100	5.8	65
	1000 R1	6.9	78
	1000 R2	7.1	80
	1000 R3	7.0	79
3,5-dichlorophenol	3.2	5.9	66
	10	7.6	85
	32	8.6	97

R₁– R₄= Replicates 1 to 4

Table 2: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2Reading (mg O2/L)	O2Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	5.5	5	2.0	42.00	-
	R2	6.2	7	2.0	36.00	-
	R3	5.4	5	2.0	40.80	-
	R4	5.7	5	2.1	43.20	-
Test Item	10	5.7	5	2.1	43.20	[7]
	100	5.8	6	1.9	39.00	4
	1000 R1	7.2	10	3.5	22.20	45
	1000 R2	7.3	10	3.6	22.20	45
	1000 R3	7.3	10	3.7	21.60	47
3,5-dichlorophenol	3.2	6.3	8	2.1	31.50	22
	10	7.4	10	5.0	14.40	64
	32	8.4	10	7.5	5.40	87

[Increase in respiration rate as compared to controls]

R₁– R₄= Replicates 1 to 4

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L.

Executive summary:

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Methods

Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of 20°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

 Results

The effect of the test item on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after
3 hours exposure was 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of
1000 mg/L.

The reference item gave a 3-Hour EC₅₀value of 7.7 mg/L, 95% confidence limits 6.1 to 9.8 mg/L.