Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: long-term

Remarks:

reproduction of Caenorhabditis elegans (Nematoda)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2016-01-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline Under GLP

Justification for type of information:

Second species chronic aquatic sediment study.

Qualifier:

according to guideline

Guideline:

other: ISO 10872 (2010): Water quality – Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Water Solubility 2 – 100µg/L
Boiling point Do not distill (Decomposes)
Log Pow 7 (25 °C / 77 °F)
Density 928 kg/m3 (20 °C / 68 °F)
Remarks None

Analytical monitoring:

not required

Remarks:

Not required in guideline

Details on sampling:

N/A

Vehicle:

yes

Remarks:

n-Hexane

Details on sediment and application:

Natural soil (loamy sand)

Ca. 24 hours before insertion of the test organisms, the respective test item amount for a stock solution (200 mg) was weighed out and dissolved in n-Hexane (25 mL). 2.5 mL of the application solution was added to 10 g sediment and thoroughly mixed. After complete evaporation of the solvent, the treated soil was given to an untreated portion of the sediment (10 g sediment dry weight). 11.3 mL M9 medium was added to obtain a water content of approx. 40 %.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

natural sediment

Limit test:

yes

Duration:

96 h

Exposure phase:

total exposure duration

Remarks:

Due to the rapid life cycle of a nematode and the assessment of reproduction and fertility this is considered a chronic endpoint

Post exposure observation period:

Mortality [X]; Growth [X]; Fertility[X]; Reproduction[X] were assessed at the end of the study

Hardness:

N/A (M9 medium used)

Test temperature:

20 ± 2 °C

pH:

5.98

Dissolved oxygen:

N/A

Salinity:

No Data

Ammonia:

No Data

Conductivity:

No Data

Nominal and measured concentrations:

1000 mg/kg sediment dry weight nominal.

Details on test conditions:

20 ± 2 °C in dark

Reference substance (positive control):

yes

Remarks:

Benzyldimethylhexadecylammonium chloride with a content of 98.3 % instead of 99.7 % was used for reference item. Result was acceptable.

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Reproduction/Mortality/Fertility/ Growth Assessed

Results with reference substance (positive control):

Acceptable acording to guideline

Validity criteria fulfilled:

yes

Conclusions:

The overall No Observed Effect Concentration (NOEC) concerning nematode mortality, fertility, growth and reproduction was ≥ 1000 mg/kg sediment dry weight.

Executive summary:

GLP guideline nematode study. Considered valid without restrictions as a second species chronic sediment test.

Reference 2

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 08 May 2013 and 11 September 2013.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to OECD test Guidelines and to GLP. Effects are considered to be genuinely substance related, however the spiking method results in high poor water concentrations far above the solubility limit and these are likely to be the cause of the effect observed. Such distribution is unlikely to occur in the environment where a more steady equilibrium is more likely. At this time no suitable alternative method for such substances exists. Study is therefore considered reliable with these restrictions.

Qualifier:

according to guideline

Guideline:

other: The OECD Guidelines for the Testing of Chemicals, “Sediment-water Lumbriculus Toxicity Test using Spiked Sediment”, OECD Guideline No. 225, October 2007

Deviations:

no

Remarks:

There were no deviations that were considered to affect the outcome or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples of the freshly prepared sediments were taken from the solvent control and all test concentrations on Day-0 for quantitative analysis.
Samples of the overlying water and sediment were taken from the solvent control and all test concentrations on Days 0 and 28 for quantitative analysis.
Analysis for the concentration of test item in the pore water of the solvent control and all test concentrations was also performed on Days 0 and 28. The pore (interstitial) water was isolated by centrifugation (10000 g at 4 °C for 30 minutes).
Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

yes

Details on sediment and application:

Experimental preparation
For the purpose of the definitive test, the test item was prepared using a preliminary solution in THF.
Prior to dosing, the air dried peat component of the artificial sediment was pre-conditioned. An amount of air dried peat (80 g) was added to 840 mL of deionized reverse osmosis water, the pH adjusted to approximately 5.5 ± 0.5 and then stirred using a magnetic stirrer for 2 days. Seven replicates were prepared, one for the control, solvent control and each test concentration. After
2 days pre-conditioning, the pH was again adjusted to approximately 6 ± 0.5. The sand (1.5 kg) and clay (0.4 kg) were mixed with the peat and then each prepared sediment was placed in a 3 liter beaker, filled with dechlorinated tap water at a ratio of 1:4 (sediment:water), aerated at a rate of approximately 3 bubbles per second and left to pre-condition for 7 days.

Following the second pre-conditioning period, the overlying water was removed from each prepared sediment and the food source (5 g each of dried nettles and α-cellulose) and test item added using a Kenwood Chef mixer to give the final test concentrations of 10, 32, 100, 320 and 1000 mg/kg with a nominal moisture content of 40 % dry weight.

Amounts of test item (2500 and 800 mg) were dissolved in THF and the volume of each adjusted to 25 mL to give the 2500 and 800 mg/25 mL solvent stock solutions. Serial dilutions were prepared in THF to give the further stock solutions of 250, 80 and 25 mg/ 25 mL. Aliquots
(20 mL) of THF and each solvent stock solution were added to approximately 50 g of artificial sediment and allowed to dry in a fume cabinet until all of the solvent had evaporated off. The control was prepared in an identical manner without the addition of test item or solvent.

During mixing of each sediment the pH was adjusted with calcium carbonate to approximately pH 6.

Each prepared sediment was then dispersed to 300 mL glass beakers (approximately 2 cm). An
8 cm layer of dechlorinated tap water was added to each vessel. A plastic disc was placed over the sediment and water poured gently onto the disc in order to avoid disturbance of the sediment. The plastic disc was then removed.

The test vessels were then left for 2 days prior to addition of the test organisms to allow settlement and equilibration of test concentrations between the sediment and water phases.

After 2 days equilibration period 10 worms were placed in each test vessel and aerated (approximately 1 bubble/second) via narrow bore glass tubes. The vessels were maintained in a temperature controlled room at approximately 21 ºC with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk periods.

Each prepared sediment was dispersed to glass beakers. Eight replicates were prepared for the solvent control and six for the control and each test group. Replicates R7 – R8 of the solvent control and replicates R5 – R6 of each test group were prepared for sacrificing on Day 0 and Day 28 for chemical analysis.

The concentration, homogeneity of the test item in the test sediment and overlying water was determined on Days 0 and 28

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

Test species
The test was carried out using adult worms of Lumbriculus variegatus derived from in-house cultures.

The worms were maintained in 40 liter glass aquaria with a substrate of shredded paper hand towels. Dechlorinated tap water was continuously passed through the tank. The culture vessels were maintained in a temperature controlled room at approximately 21 ºC. The lighting cycle was controlled to give a 16 hour light and 8 hour darkness cycle with 20 minute dawn and dusk transition periods. The cultures were gently aerated so as not to disturb the substrate, through narrow bone glass tubes.

The worms were fed with Tetramin® flake food at approximately 30 mL of a 5 g/100 mL dispersion every day. The Tetramin® flake food was prepared as a suspension in water and an appropriate volume added to the overlying water.

The diet and diluent were considered not to contain any contamination that would affect the integrity or outcome of the test.

Approximately 10 to 14 days prior to the start of the test, sufficient numbers of worms were dissected in the median body region with a scalpel. The posterior ends were placed in culture medium with a small amount of culturing substrate and left to regenerate new heads. This was conducted to synchronize the age of the worms.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Nominal and measured concentrations:

Range-finding test: 1.0, 10, 100 and 1000 mg/kg (dry weight of sediment)
Definitive test: 10, 32, 100, 320 and 1000 mg/kg (dry weight of sediment).

Details on test conditions:

Test Water
The test water used for the range-finding and definitive tests was the same as that used to maintain the stock cultures. Laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

Formulated Sediment
A defined formulated sediment was used in the following composition:
Industrial quartz sand: 75 % w/w
Kaolinite clay: 20 % w/w
Sphagnum peat, ground and air dried: 4 % w/w
Powdered stinging nettle leaves: 0.25 % w/w (in addition to the components above)
α cellulose: 0.25 %
Calcium carbonate was added to bring the pH range within 7.0 ± 0.5.

Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.

In the range-finding test Lumbriculus variegatus were exposed to a series of nominal test concentrations of 1.0, 10, 100 and 1000 mg/kg (dry weight of sediment). The test item was prepared using a preliminary solution in tetrahydrofuran (THF).

Prior to dosing, the air dried peat component of the artificial sediment was pre-conditioned. An amount of air dried peat (0.02 kg) was added to 200 mL of deionized reverse osmosis water, the pH adjusted to 5.5 ± 0.5 and then stirred using a magnetic stirrer for 2 days. Six replicates were prepared, one for the control, solvent control and each test concentration. After 2 days pre-conditioning, the pH was again adjusted to approximately 6 ± 0.5. The sand (0.375 kg) and clay (0.1 kg) were mixed with the peat and then each prepared sediment was placed in a 3 liter beaker, filled with dechlorinated tap water at a ratio of 1:4 (sediment:water), aerated at a rate of approximately 3 bubbles per second and left to pre-condition for 7 days.

Following the second pre-conditioning period, the overlying water was removed from each prepared sediment and the food source (1.25 g each of dried nettles and α-cellulose) and test item added using a Kenwood Chef mixer to give the final test concentrations of 1.0, 10, 100 and
1000 mg/kg with a nominal moisture content of 40% dry weight.

An amount of test item (1000 mg) was dissolved in tetrahydrofuran (THF) and the volume adjusted to 10 mL to give a 1000 mg/10 mL solvent stock solution. Serial dilutions were prepared in THF to give the further stock solutions of 100, 10 and 1.0 mg/ 10 mL. Aliquots (5.0 mL) of THF and each solvent stock solution were added to approximately 10 g of artificial sediment and allowed to dry in a fume cabinet until all of the solvent had evaporated off. The control was prepared in an identical manner without the addition of test item or solvent.

During mixing of each sediment the pH was adjusted with calcium carbonate to approximately pH 6.

Each prepared sediment was then dispensed to 300 mL glass beakers (approximately 2 cm). An 8 cm layer of dechlorinated tap water was added to each vessel. A plastic disc was placed over the sediment and water poured gently onto the disc in order to avoid disturbance of the sediment. The plastic disc was then removed.

The test vessels were then left for 2 days prior to addition of the test organisms to allow settlement and equilibration of test concentrations between the sediment and water phases.

After 2 days equilibration period 10 worms were placed in each test vessel and the test vessels were aerated (approximately 2 bubbles/second) via narrow bore glass tubes. The vessels were maintained in a temperature controlled room at approximately 21 ºC with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk periods.

Observations on the general condition of the worms in each vessel were made daily.

The measured end point for the test was the number of live worms on Day 28. For each test vessel, a sediment slurry was formed, poured into a shallow tray and the worms removed with tweezers and counted.

The dry weight of surviving worms for each test replicate was determined on Day 28. The worms were dried at approximately 60°C for approximately 3 days.


Definitive Test
Based on the results of the range-finding test the following test concentration range was assigned to the definitive test; 10, 32, 100, 320 and 1000 mg/kg (dry weight of sediment).


Exposure Conditions
As in the range-finding test, 300 mL glass beakers were used. After the 2-Day equilibration period 10 worms were placed in each test (Replicates R1-R4) and control and solvent control vessel (Replicates R1 – R6) and maintained in a temperature controlled room at approximately 21 ºC with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk periods.

Losses of overlying water due to evaporation were adjusted daily, if necessary, by the addition of deionized water.

Observations on the general condition of the worms in each vessel were made daily.

The measured end-point for the test was the number of live worms. The number of live worms in each replicate was determined on Day 28.

For each replicate test vessel, a sediment slurry was formed, poured into a shallow tray and the worms removed with tweezers and counted.

The dry weight of surviving worms for replicates R1 – R6 of the control and solvent control and replicates R1 – R4 for each test concentration were determined at the end of the test. The worms were dried at approximately 60 °C for approximately 5 days.

The control and solvent control groups were maintained under identical conditions but not exposed to the test item.

Reference substance (positive control):

yes

Remarks:

pentachlorophenol sodium salt (PCP-Na salt)

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

69 other: mg/kg

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

5.1 other: mg/kg

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

21 other: mg/kg

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Range-finding test
On Day 28 there were comparable numbers of worms recorded in the control, solvent control, 1.0 and 10 mg/kg test concentrations. However fewer surviving worms were observed in the 100 and 1000 mg/kg test concentrations.

The organic carbon content of the sediment was determined to be 1.83 %.

Based on this information test concentrations of 10, 32, 100, 320 and 1000 mg/kg (dry weight of sediment) were selected for the definitive test.


Definitive test
Analysis of the numbers of surviving worms at the end of the test by the Maximum Likelihood-Probit method (Finney, 1977) using the ToxCalc computer software package (ToxCalc, 1999) based on the Day 0 measured test concentrations showed the 28 day EC50 (reproduction) was 69 mg/kg.

The EC10 has not been reported due to the unsuitable nature of the data. Statistical analysis of the numbers of surviving worms at the end of the test was carried out for the control, solvent control and all test concentrations using one way analysis of variance incorporating Bartlett’s Test for homogeneity of variance (Sokal and Rohlf, 1981). There were no significant differences (P≥0.05) between the solvent control and the control and 5.1 mg/kg test groups in terms of the number of surviving worms at the end of the test. However the 21, 81, 292 and 833 mg/kg test groups were significantly different (P<0.05) from the solvent control in terms of the number of surviving worms at the end of the test. Therefore the No Observed Effect Concentration (NOEC) based on survival was 5.1 mg/kg.

The Lowest Observed Effect Concentration (LOEC) based on survival was 21 mg/kg.

Observations on the test organism
Sub-lethal effects of exposure to the test item were observed in the 100, 320 and 1000 mg/kg test groups. These included fewer fecal pellets than in the control and solvent control, sediment avoidance and erratic movement.

Observations on the Test Sediment
No abnormalities were observed in the sediment layer or overlying water.

Statistical analysis of the worm weight data at the end of the test was carried out for the control, solvent control and all test concentrations using one way analysis of variance incorporating Bartlett’s Test for homogeneity of variance (Sokal and Rohlf, 1981). There were no significant differences (P≥0.05) between the solvent control and the control, 5.1 and 21 mg/kg test groups. However the 81, 292 and 833 mg/kg test groups were significantly different (P<0.05) from the solvent control in terms of the weight of surviving worms at the end of the test.

Results with reference substance (positive control):

A positive control (Harlan Study Number: 41302621) used pentachlorophenol sodium salt (PCP-Na salt) as the reference item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/kg (dry weight of sediment).

Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the numbers of surviving worms at the end of the test by the trimmed Spearman-Karber method (Hamilton et al 1977 ) based on nominal test concentrations gave a 28-day EC50 (reproduction) of 26 mg/kg with 95 % confidence limits of 22 - 31 mg/kg.
The No Observed Effect Concentration (NOEC) was 10 mg/kg.
The Lowest Observed Effect Concentration (LOEC) was 32 mg/kg.

The observed EC50 was within the 4.0 to 37.9 mg/kg range given in the Test Guideline.

Reported statistics and error estimates:

Analysis of Numbers of Surviving Worm
Analysis of the number of surviving worms obtained at termination of the test from the control, solvent control, 5.1, 21, 81, 292 and 833 mg/kg test groups were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981). Data was transformed by log10 to ensure homogeneity of variance.

The number of surviving worms of the control, solvent control, 5.1, 21, 81, 292 and 833 mg/kg test groups were used for this analysis. The analysis was performed using the individual number of surviving worms per replicate for each test group.

No significant differences (P≥0.05) were found between the solvent control, control and 5.1 mg/kg test groups using the above methods of statistical analysis. However significant differences (P<0.05) were found between the solvent control and the 21, 81, 292 and 833 mg/kg test groups.

Analysis of Worm Dry Weight
Analysis of the worm dry weight data obtained at termination of the test from the control, solvent control, 5.1, 21, 81, 292 and 833 mg/kg and test groups were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf, 1981) .

The worm weight data of the control, solvent control, 5.1, 21, 81, 292 and 833 mg/kg test groups were used for this analysis. The data was transformed by log10 function to ensure homogeneity. The analysis was performed using the individual mean worm weight values, from each replicate for each test group.

No significant differences (P≥0.05) were found between the solvent control and the control, 5.1 and 21 mg/kg test groups using the above methods of statistical analysis. However, significant differences (P<0.05) were found between the control and the 81, 292 and 833 mg/kg test groups.

Results Number of Surviving Worms in the Range-Finding Test

Nominal Concentration (mg/kg)	Number of Surviving Worms
	Day 28
Control	30
Solvent Control	33
1.0	33
10	32
100	21
1000	14

Worm Weight Data for the Range-Finding Test

Nominal Concentration (mg/kg)	Mean Individual Worm Dry Weight (mg)
	Day 28
Control	1.82
Solvent Control	1.12
1.0	1.98
10	1.19
100	1.53
1000	0.26

Number of Surviving Worms in the Definitive Test after 28 Days

Day 0 Measured Concentration (mg/kg)	Number of Surviving Worms
	Day 28
	R1	R2	R3	R4	R5	R6
Control	26	34	26	32	29	29
Solvent Control	29	31	25	29	35	29
5.1	26	29	30	30	-	-
21	13	14	14	18	-	-
81	10	10	10	10	-	-
292	8	10	10	9	-	-
833	10	9	8	10	-	-

R₁– R₆= Replicates 1 to 6

Worm Weight in the Definitive Test after 28 Days

Day 0 Measured Concentration (mg/kg)	Mean Individual Worm Dry Weight (mg)
	Replicate
	1	2	3	4	5	6
Control	1.20	1.20	2.00	1.40	1.70	1.70
Solvent Control	1.10	1.50	1.80	1.70	1.20	1.80
5.1	0.92	1.40	1.60	1.30	-	-
21	0.68	1.70	0.61	0.45	-	-
81	1.50	0.23	0.37	0.32	-	-
292	0.20	0.15	0.22	0.74	-	-
833	0.46	0.34	0.35	0.27	-	-

Analysis of Numbers of Surviving Worms

Nominal Concentration (mg/kg)	Mean Number of Surviving Worms
ControlR1– R6	Mean	29
	Standard Deviation	3.2
Solvent Control R1– R6	Mean	30
	Standard Deviation	3.3
5.1 R1– R4	Mean	29
	Standard Deviation	1.9
21 R1– R4	Mean	15
	Standard Deviation	2.2
81 R1– R4	Mean	10
	Standard Deviation	0
292 R1– R4	Mean	9.2
	Standard Deviation	1.0
833 R1– R4	Mean	9.2
	Standard Deviation	1.0

R₁– R₆= Replicates 1 to 6

Analysis of Worm Dry Weight

Day 0 Measured Concentration (mg/kg)	Mean Individual Worm Weight (mg)
ControlR1– R6	Mean	1.5
	Standard Deviation	0.32
Solvent Control R1– R6	Mean	1.55
	Standard Deviation	0.31
5.1 R1– R4	Mean	1.3
	Standard Deviation	0.37
21 R1– R4	Mean	0.86
	Standard Deviation	0.57
81 R1– R4	Mean	0.61
	Standard Deviation	0.60
292 R1– R4	Mean	0.33
	Standard Deviation	0.28
833 R1– R4	Mean	0.36
	Standard Deviation	0.79

R₁– R₆= Replicates 1 to 6

pH Values of Prepared Test Sediments

Day 0 Measured Concentration (mg/kg)	pH Values of Prepared Test Sediment
Control	6.2
Solvent Control	6.0
5.1	6.1
21	6.2
81	6.3
292	6.1
833	6.1

Physico-chemical Measurements

Day 0 Measured Concentration (mg/kg)	Day 0				Day 7				Day 14				Day 21				Day 28
	pH	mg O2/L	%ASV*	T°C	pH	mg O2/L	% ASV*	T°C	pH	mg O2/L	% ASV*	T°C	pH	mg O2/L	% ASV*	TºC	pH	mg O2/L	% ASV*	TºC
Control	8.2	8.5	96	21	8.3	8.5	96	21	8.2	7.9	89	21	8.2	8.1	91	21	8.3	8.5	93	20
Solvent control	8.3	8.7	98	21	8.3	8.6	97	21	8.3	8.2	92	21	8.3	8.3	93	21	8.4	8.5	93	20
5.1	8.3	8.7	98	21	8.2	8.5	96	21	8.2	8.2	92	21	8.2	8.1	91	21	8.3	8.1	88	20
21	8.2	8.7	98	21	8.2	8.4	94	21	8.2	8.2	92	21	8.2	8.1	91	21	8.3	8.2	90	20
81	8.2	8.7	98	21	8.2	8.5	96	21	8.3	8.2	92	21	8.3	8.2	92	21	8.4	8.4	92	20
292	8.3	8.7	98	21	8.3	8.7	98	21	8.3	8.4	94	21	8.3	8.4	92	20	8.4	8.4	92	20
833	8.2	8.6	97	21	8.2	8.5	96	21	8.3	8.4	94	21	8.3	8.5	93	20	8.4	8.5	93	20

*ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

Room Temperature and Light Intensity Records

Day	Room Temperature (°C)		Light Intensity (Lux)
	Maximum	Minimum
0	21	21	387
1	23	20	390
2	23	20	386
3	22	21	387
4	22	20	389
5	23	21	379
6	22	21	383
7	23	21	385
8	22	21	389
9	22	21	386
10	22	21	389
11	23	22	386
12	23	21	384
13	23	21	385
14	23	21	386
15	23	21	383
16	22	21	384
17	22	21	382
18	22	21	383
19	23	21	382
20	23	21	356
21	23	21	371
22	23	21	363
23	23	21	369
24	23	21	343
25	23	21	378
26	23	21	391
27	23	21	367
28	22	21	270

Ammonia Determination

Day	Ammonia Concentration (NH3mg/L)
	Control	Solvent Control	10 mg/kg	32 mg/kg	100 mg/kg	320 mg/kg	1000 mg/kg
0	0.00071	0.00140	0.00059	0.00018	0.00030	0.00000	0.00024
3	0.01591	0.00900	0.00689	0.00808	0.00788	0.00508	0.00518
5	0.02230	0.01869	0.02208	0.02179	0.01111	0.00403	0.01030
7	0.00784	0.01057	0.01384	0.01657	0.01307	0.01243	0.00944
10	0.01045	0.02891	0.01281	0.00790	0.01214	0.02791	0.02031
12	0.02034	0.00855	0.00663	0.00529	0.01465	0.03577	0.02927
14	0.00238	0.01479	0.01675	0.00787	0.00832	0.01111	0.03813
17	0.01097	0.20988	0.01580	0.00396	0.00290	0.00520	0.00682
19	0.00043	0.2082	0.00588	0.00504	0.00134	0.00218	0.00109
21	0.00103	0.02002	0.00147	0.00169	0.00684	0.00546	0.00824
24	0.00800	0.01600	0.01200	0.00600	0.00200	0.00100	0.00100
26	0.00100	0.02100	0.02800	0.00300	0.00200	0.00200	0.00100
28	0.03300	0.02500	0.00400	0.00500	0.00000	0.00000	0.00000

Physico-chemical Measurements

Temperature was maintained at approximately 21 ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Room temperature remained at approximately 22 °C throughout the test and the light intensity was 270 to 391 lux.

The water hardness for the solvent control was determined to be 160 and 156 mg/L as CaCO₃on Days 0 and 28 respectively, and 164 and 152 mg/L as CaCO₃for the 1000 mg/kg test concentration on Days 0 and 28 respectively.

 

The conductivity for the solvent control was determined to be 631 and 562 µS/cm on Days 0 and 28 respectively, and 582 and 557 µS/cm for the 1000 mg/kg test concentration on Days 0 and 28 respectively.

 

The ammonia concentrations were determined to range from 0.0086 to 0.0290 mg/L as NH₃for the solvent control and from 0.0000 to 0.0380 mg/L as NH₃for the 1000 mg/kg test concentration.

The Total Organic Carbon (TOC) of the sediment was determined to be 2 % of the sediment dry weight.

Verification of Test Concentrations

Analysis of the sediment on Day 0 of the test showed the measured concentrations to range from 51% to 91% of nominal. The 10 and 32 mg/L samples were reanalyzed due to measured concentrations being low compared to nominal, however, similarly low measured concentrations were obtained. Analysis of the overlying water on Day 0 showed measured concentrations to range from less than the limit of quantification, determined to be 0.0058 mg/L, to 0.0076 mg/L. Analysis of the interstitial water on Day 0 of the test showed measured concentrations to range from less than the limit of quantification, determined to be 0.029 mg/L, to 63.7 mg/L.

 

Analysis of the sediment on Day 28 of the test showed the measured concentrations to range from 12% to 81% of nominal. Analysis of the overlying water on Day 28 showed the measured concentrations to be less than the limit of quantification. Analysis of the interstitial water on Day 28 of the test showed measured concentrations to range from less than the limit of quantification to 11.7 mg/L.

 

On Day 0 there was a lower percentage of nominal recovered from the lower concentrations in both original and duplicate samples when compared to the higher concentrations, this could have been due to the test item binding to the sediment matrix or possibly due to the volatile nature of the test item (EPIWIN estimated Henry’s Law Constant: 2.363 x 10³atm.m³.mol^-1). These factors could also be used to explain the decline seen after 28 days.

 

Validation Criteria

The dissolved oxygen concentration was at least 30% Air Saturation Value (ASV) at the end of the test and the pH of the overlying water was in the range pH 6 to 9.

The water temperature did not vary by more than ± 1.0°C between replicates at any one time.

The number of worms in the controls at the end of the test increased by a factor of more than 1.8.

Validity criteria fulfilled:

yes

Conclusions:

The Day 28 EC50 (reproduction) based on the Day 0 measured test concentrations was 69 mg/kg. The EC10 has not been reported due to the unsuitable nature of the data. 95% confidence limits could not be determined from the data. The No Observed Effect Concentration was 5.1 mg/kg and the Lowest Observed Effect Concentration was 21 mg/kg.

Executive summary:

Introduction

A study was performed to assess the toxicity of the test item on reproduction to the sediment-dwelling oligochaete, Lumbriculus variegatus. The method was based on the method of The OECD Guidelines for the Testing of Chemicals, “Sediment-water Lumbriculus Toxicity Test using Spiked Sediment”, OECD Guideline No. 225, October 2007

  

Methods…….

Following a preliminary range-finding test, worms of Lumbriculus variegatus (4 to 6 replicates of 40 to 60 worms) were exposed to formulated sediment spiked with test item at concentrations of 10, 32, 100, 320 and 1000 mg/kg (dry weight of sediment) for a period of 28 days. The numbers of worms and the dry weight data of these worms were recorded at the end of the test.

 

Further replicates were prepared for each test group and sacrificed on Days 0 and 28 for chemical analysis of the sediment, interstitial and overlying water.

 

Results

TheDay 28 EC₅₀ (reproduction) based on the Day 0 measured test concentrations was 69 mg/kg, 95% confidence limits could not be statistically determined from the data. The EC₁₀has not been reported due to the unsuitability of the data. The No Observed Effect Concentration was 5.1 mg/kg and the Lowest Observed Effect Concentration was 21 mg/kg.

 

Analysis of the sediment on Day 0 of the test showed the measured concentrations to range from 51% to 91% of nominal. The 10 and 32 mg/L samples were reanalyzed due to measured concentrations being low compared to nominal, however, similarly low measured concentrations were obtained. Analysis of the overlying water on Day 0 showed measured concentrations to range from less than the limit of quantification, determined to be 0.0058 mg/L, to 0.0076 mg/L. Analysis of the interstitial water on Day 0 of the test showed measured concentrations to range from less than the limit of quantification, determined to be 0.029 mg/L, to 63.7 mg/L.

 

Analysis of the sediment on Day 28 of the test showed the measured concentrations to range from 12% to 81% of nominal. Analysis of the overlying water on Day 28 showed the measured concentrations to be less than the limit of quantification. Analysis of the interstitial water on Day 28 of the test showed measured concentrations to range from less than the limit of quantification to 11.7 mg/L.

Conclusion

Study was conducted according to the appropriate test guideline with GLP accreditation and a suitable analytical method and certificate of analysis. However sufficient adaption for test chemical properties did not take place. This lead to pore water concentrations thousands of times higher than the water solubility limit. Effects could therefore be a product of the instant spiking method that in the environment occurs gradually via spiking at or under the solubility limit not via a solvent.

Description of key information

Two chronic sediment studies are available.

OECD 225 guideline study is not considered optimal for extremely poorly water soluble substances due to the high pore water concentrations measured and the lack of sediment equilibrium. A more realistic indication of sediment effects is at this time not possible data is considered reliable with this restriction. The partitioning of this substance to sediment was 97%. Similar adsorbtion to sludge has been demonstrated in a sewage treatment simulation test and hence in reality the test substance is unable to pass the water treatment plant. Meaning that sediment exposure is highly unlikely due to the industrial use of the substance and treated effluent only being released.

An ISO 10872 (2010) sediment test with nematodes is also available and no effects to any endpoint were observed up to 1000 mg/kg sediment dw.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

5.1 mg/kg sediment dw

Additional information

The 28 Day EC₅₀(worst case end point reproduction) based on the Day 0 measured test concentrations was 69 mg/kg, 95% confidence limits could not be statistically determined from the data. The EC₁₀has not been reported due to the unsuitability of the data. The No Observed Effect Concentration was 5.1 mg/kg and the Lowest Observed Effect Concentration was 21 mg/kg.

 

Analysis of the sediment on Day 0 of the test showed the measured concentrations to range from 51% to 91% of nominal. The 10 and 32 mg/L samples were re-analyzed due to measured concentrations being low compared to nominal, however, similarly low measured concentrations were obtained. Analysis of the overlying water on Day 0 showed measured concentrations to range from less than the limit of quantification, determined to be 0.0058 mg/L, to 0.0076 mg/L. Analysis of the interstitial water on Day 0 of the test showed measured concentrations to range from less than the limit of quantification, determined to be 0.029 mg/L, to 63.7 mg/L.

 

Analysis of the sediment on Day 28 of the test showed the measured concentrations to range from 12% to 81% of nominal. Analysis of the overlying water on Day 28 showed the measured concentrations to be less than the limit of quantification. Analysis of the interstitial water on Day 28 of the test showed measured concentrations to range from less than the limit of quantification to 11.7 mg/L.

The guideline was followed and the study was conducted in a reliable manner for non difficult substances. However due to the extremely poor solubility of the test chemical in question and the indication of pore water concentrations way in excess of the solubility limit. It appears that the instantaneous spiking of the sediment and short resting period was not sufficient to allow steady state and even distribution in the sediment. Sediment exposure can only ever occur in reality at a maximum of 93 µg/L via water with significantly longer resting periods.

Chronic nematode test ISO 10872 (2010) shows no effects to any endpoint up to 1000 mg/kg dw