1,6-Hexanediamine, N1,N6-bis(1,2,2-trimethylpropyl)-

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:

27 July to 18 August 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Followed existing guideline and GLP requirements; formulations not analyzed, a few dissolved O2 values were below those recommended in test guidelines, but results based on oxygen consumption rates over the linear portion of the oxygen consumption curves

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

No analysis of formulations, some out of limit dissolved oxygen concentrations

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

- Concentrations: Range find: nominal concentrations of 10, 100 and 1000 mg/l
Definitive test: nominal concentrations of 100, 180, 320, 560, and 1000 mg/l
5 replicates per concentration were prepared
- Sampling method: As each vessel reached 3 hours contact time, an aliquot was removed from the conical vessel and poured into the measureing vessel for rate of respiration monitoring.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solutions were prepared by separately dispersing the test item (50, 90, 160, 280 and 500 mg, five replicates of each) into approximately 200 ml deionized reverse osmosis water and subjecting to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximize the dissolved test article concentration. Test vessels were shielded from light during mixing. Synthetic sewage (16 ml), activated sludge, (250 ml) and water were added to give a final volume of 500 ml to give final concentrations of 100, 180, 320, 560, and 1000 mg/l. pH of test item dispersions were measured after stirring and prior to the addition of inoculum and adjusted to between pH 7.0 and 8.0.
- Controls: Control group prepared as others but not exposed to test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Observations on test preparations throughout the test period in the definitive test:
Control replicates: 0 hour (prior to activated sludge addition) pale yellow brown dispersion
30 min contact time dark brown dispersion
3 hour contact time dark brown dispersion

100 mg/l 0 hour Pale yellow/brown dispersion with a few oily globules of test item on surface and dispersed throughout
30 min contact time Dark brown dispersion with a few oily globules of test item on
surface and dispersed throughout
3 hour contact time Dark brown dispersion with a few oily globules of test item on
surface and dispersed throughout
180 mg/l 0 hour Pale yellow/brown dispersion with a few oily globules of test item on surface and dispersed throughout
30 min contact time Dark brown dispersion with a few oily globules of test item on
surface and dispersed throughout
3 hour contact time Dark brown dispersion with a few oily globules of test item on
surface and dispersed throughout

320 mg/l 0 hour Pale yellow/brown dispersion with a few oily globules of test item on surface and dispersed throughout
30 min contact time Dark brown dispersion with a few oily globules of test item on
surface and dispersed throughout
3 hour contact time Dark brown dispersion with a few oily globules of test item on
surface and dispersed throughout
560 mg/l 0 hour Pale yellow/brown dispersion with a few oily globules of test item on surface and dispersed throughout
30 min contact time Dark brown dispersion with a few oily globules of test item on
surface and dispersed throughout
3 hour contact time Dark brown dispersion with a few oily globules of test item on
surface and dispersed throughout



1000 mg/l 0 hour Pale yellow/brown dispersion with a few oily globules of test item on surface and dispersed throughout
30 min contact time Dark brown dispersion with a few oily globules of test item on
surface and dispersed throughout
3 hour contact time Dark brown dispersion with a few oily globules of test item on
surface and dispersed throughout


3,5 dichlorophenol 0 hour Pale yellow/brown dispersion with no undissolbed reference (all concentrations) material visible
30 min contact time Dark brown dispersion with no visible reference material
visible
3 hour contact time Dark brown dispersion with no visible reference material
visible

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: Mixed population of activated sewage sludge microorganisms was obtained on 27 July for the range find test and on 17 August for the definitive test from aeration stage of the Severn Trent Water Plv sewage treatment plant at Loughborough, Leicestershire, UK, which treats primarily domestic sewage.
- Method of cultivation: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at about 21 degrees C overnight prior to use in the test. On the day of collection, the activated sewage sludge (10 liters) was fed synthetic sewage sludge (500 ml). The pH of the sample on the day of the test was 7.8. Determination of suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sludge by suctin 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 was continued for 3 minutes. The filter paper was then dried in an oven at about 105 degrees C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was obtained. Suspended solids concentration was equal to 3.2 g/l prior to use.
- Preparation of inoculum for exposure: At time 0, 16 ml of synthetic sewage was diluted to 250 ml with water and 250 ml of inoculum were added to 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 about 0.5 to 1 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 material vessels with appropriate amounts of reference item added. Two additional control vessels were then prepared prior to the test item vessels being prepared. Finally, 2 further controls were prepared.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Observations made at time 0 of contact, at 30 minutes and 3 hours of contact.

Hardness:

Test water used for range finder and definitive tests was deionized reverse osmosis water containing less than 1 mg/l Dissolved Organic Carbon.

Test temperature:

20 +/- 2 degrees C in the preliminary and definitive tests

pH:

pH of test system dispersions were measured after stirring and prior to administration of inoculum and adjusted to between pH 7.0 and 8.0

pH of control, reference item and test item preparations in the test were measured at times 0, and prior to measurement of oxygen consumption rate after 3 minutes contact time. pH ranges at start and end of definitive test ranged as follows:
Control 0 hours 7.4 to 7.7 3 hours 7.6 to 8.1
100 mg/l 0 hours 7.6 to 7.7 3 hours 8.0 to 8.2
180 mg/l 0 hours 7.7 to 7.9 3 hours 7.9 to 8.1
320 mg/l 0 hours 7.6 to 8.0 3 hours 8.1 to 8.2
560 mg/l 0 hours 7.9 to 8.1 3 hours 8.4 to 8.7
1000 mg/l 0 hours 7.9 to 8.1 3 hours 9.5 to 9.6
3,5 DCP 0 hours 7.6 to 7.7 3 hours 8.2 to 8.3
(all concentrations)

Dissolved oxygen:

Linear portion of of oxygen consumption trace between 7.0 mg O2/l and 2.0 mg/O2/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 ml conical flask and 250 ml darkened glass Biological Oxygen Demand bottle
- Aeration: clean, oil-free compressed air at 0.5 to 1 liter from time 0 to 3 hours contact time
- No. of vessels per concentration (replicates): 5 for treatment concentrations
- No. of vessels per control (replicates): 5 per control group

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized reverse osmosis water
- Total organic carbon: dilution water contained less than 1 mg/l dissolved organic carbon
- Metals: analyzed at regular intervals by the lab
- Pesticides: analyzed at regular intervals by the lab
- Chlorine: analyzed at regular intervals by the lab
- Alkalinity: analyzed at regular intervals by the lab
- Ca/mg ratio: analyzed at regular intervals by the lab
- Conductivity: analyzed at regular intervals by the lab
- Other: Synthetic Sewage added to test vessels as a respiratory substrate (1 liter contained 16 g Peptone, 11 g Meat Extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSo4.7H2O, 2.8 g K2HPO4, with the aid of ultrasonication).


OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: N/A
- Light intensity: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Rate of respiration measured with Yellow Springs dissolved oxygen meter fitted with a BOD probe, after 3 hours contact, over a 10 minute period; dissolved oxygen content in flasks at 30 minutes contact, pH at 0 hours contact and prior to measurement of oxygen consumption rate after 3 hours contact.

TEST CONCENTRATIONS
- Range finding study: 10, 100, and 1000 mg/l (3 replicates)
- Test concentrations: 100, 180, 320, 560, and 1000 mg/l (5 replicates)
- Results used to determine the conditions for the definitive study: percent inhibition

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

ca. 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 460 to 550 mg/l 95% CL

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 180 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 3 hr EC50 for 3,5 dichlorophenol was 8.9 mg/l with 95% CL 7.2 to 11 mg/l

Reported statistics and error estimates:

The respiration rate, R, expressed in milligrams of oxygen per liter per hour (mg O2/l/h) was calculated from the linear part of the recorded oxygen decrease graph according to the equation: R = (Q1-Q2)/ Delta t x 60. Q1 is the oxygen concentration at the beginning of the selected section of the linear phase (mg/l); Q2 is the oxygen concentration at the end of the selected section of the linear phase (mg/l); and Delta t is the time interval between the two measurements in minutes.
The specific respiration rate, Rs, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour (mg/g/h) was deduced from the equation: Rs = R/SS, where SS is the concentration of suspended solids in the test mixture (g/l).
The percentage inhibition was calculated according to the following: % inhibition = [1-(R/Rbc)] x 100 where Rbc is the mean respiration rate of the blank controls.

The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit software package (IDBS) and the EC15, EC20, EC50 and EC80 values determined by the equation for the fitted line. 95% confidence limits were determined for the EC50 values using the method of Litchfield and Wilcoxon, 1949. One way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sodal and Rohlf, 1981) and Dunnett’s multiple comparison procedure fro comparing several treatments with a control(Dunnett, 1955) was carried out on the oxygen consumption data after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999-2001).

	SD10		3,5 dichlorophenol
	ECx (3hrs) (mg/l)	95% Confidence Limits (mg/l)	ECx (3hrs) (mg/l)	95% Confidence Limits (mg/l)
EC20	350	-	2.9	-
EC50	500	460 – 550	8.9	7.2 - 11
EC80	710	-	27	-
NOEC	180	-	-	-

The coefficient of variation of oxygen uptake in the control vessels was 7% and the specific respiration rate of the controls was 26.76 mg oxygen per gram dry weight of sludge per hour. The validation criteria were therefore satisfied, as were the validation criteria of the reference article EC50 values. Oxygen Consumption Rates and Percentage Inhibition Values after 2 hours Contact Time in the Definitive Test

Nominal concentration (mg/l)	Initial O2 (mg O2/l)	Time measured (minutes)	Final O2 (mg O2/l)	O2 Consumption Rates (mg O2/l/hour)	% inhibition
Control R1 R2 R3 R4 R5	4.2 4.8 5.2 5.0 6.6 5.3	3 4 4 4 5 5	1.9 2.0 2.2 2.1 3.0 2.2	46.00 42.00 45.00 43.50 43.20 37.20	- - - - - -
Test Article 100R1 100R2 100R3 100R4 100R5 180R1 180R2 180R3 180R4 180R5 320R1 320R2 320R3 320R4 320R5 560R1 560R2 560R3 560R4 560R5 1000R1 1000R2 1000R3 1000R4 1000R5	3.9 3.7 4.4 4.4 3.8 3.8 4.2 4.3 3.9 2.2 3.8 3.6 5.4 3.7 4.2 6.7 6.8 5.7 6.0 7.0 9.1 8.4 8.7 8.6 8.0	3 3 3 3 3 3 4 4 3 1 3 3 7 3 4 10 10 10 4 5 5 5 5 5 5	1.8 1.5 2.0 2.3 1.7 1.7 1.5 1.6 1.8 1.5 1.6 1.7 2.3 1.8 1.8 4.6 4.6 2.8 4.4 5.6 9.1 8.3 8.5 8.5 7.5	42.00 44.00 48.00 42.00 42.00 42.00 40.50 40.50 42.00 42.00 44.00 38.00 26.57 38.00 36.00 12.60 13.20 17.40 24.00 16.80 0.00 1.20 2.40 1.20 6.00	2 [3] [12] [2] 2 2 5 5 2 2 [3] 11 38 11 16 71 69 59 44 61 100 97 94 97 86
3,5 dichloro-phenol 3.2 10 32	6.5 7.1 7.7	7 9 10	2.5 4.4 6.4	34.29 18.00 7.80	20 58 82

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test article on the respiration of activated sewage sludge gave a 3-hour EC50 value of 500 mg/l, 95% confidence limit 460-550 mg/l. The No Observed Effect Concentration after 3 hours exposure was 180 mg/l.

Executive summary:

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous solution of test substance at concentrations of 100, 180, 320, 560, and 1000 mg/l (5 replicates) for a period of 3 hours at a temperature of 20 +/- degrees 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 the control and reference substance (3,5 -dichlorophenol) data. The reference item gave a 3 -hr EC50 value of 8.9 mg/l, 95% confidence limits 7.2 to 11 mg/l. The test substance gave a 3 -hour EC50 value of 500 mg/l, 95% confidence limits of 460 to 550 mg/l. The No Observed Effect Concentration (NOEC) was 180 mg/l after 3 hour exposure. The test followed OECD Guideline No. 209 "Activated sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation )"

Description of key information

A respiration inhibition test according to OECD guideline N°209 and GLP was used by exposing activated sludge to different concnetrations of the test item during 3 hours. The test substance gave a 3 hours EC50 value of 500 mg/L (nominal concentration) and a NOEC value of 180 mg/L (nominal concentration).

Key value for chemical safety assessment

EC50 for microorganisms:

500 mg/L

EC10 or NOEC for microorganisms:

180 mg/L