Amines, N-C10-16-alkyltrimethylenedi-, reaction products with chloroacetic acid

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014-10-07 to 2015-05-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)

Version / remarks:

adopted 13 April, 2004

Deviations:

yes

Remarks:

Only minor deviations concerning light intensity and food. The deviations are assumed to be without influence on the integrity and quality of the study and the interpretation of the results.

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Analytical monitoring:

yes

Details on sampling:

SEDIMENT
- Concentrations: 46.9, 93.8, 187.5, 375, 750, and 1500 mg/kg dry sediment (after an equilibration period of 48 hours after addition of the test item) from one vessel per treatment, 46.9, 375 and 1500 mg/kg dry sediment (test end), 375 mg/kg dry sediment (test day 7 and 14)

OVERLYING WATER
- Concentrations: 375 and 1500 mg/kg dry sediment (test start and test end), 46.9 mg/kg sediment dry weight (test end)

Vehicle:

no

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
Test item was applied via a stock solution of the test item dissolved in deionised water. The respective portion of sediment (adjusted to 20 – 40 % water content to avoid waterlogging during distribution) was filled in the test vessel. The quantity of stock solution required to obtain the desired concentration was added to the sediment. Subsequently, the sediment was mixed thoroughly with the applied stock solution by hand mixing. The overlaying water was filled in the vessels carefully to avoid sediment raise.
- Equilibration time: 2 days
- Controls: formulated sediment only

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Common name: harlequin fly
- Source: Bayer Crop Science AG, 40789 Monheim, Germany. Specimens used in the test were bred in the laboratory of the Fraunhofer IME.
- Breeding conditions: The breeding conditions follow SOP V4-601/03. On a layer of diatomaceous earth purified tap water was used. The dipterans were fed daily with powder of TetraMin® Hauptfutter (Tetra Werke, Melle, Germany).
- Handling of egg masses and larvae: Four to five days before adding the test organisms to the test vessels, egg masses were taken from the cultures and placed in small aerated vessels with test water at about 20 °C. First instar larvae (2 days post hatching) were used in the test.
- Age of animals at beginning of exposure: 2 days post-hatching

- Feeding during test: yes. Due to the strong adsorbing character of the test item, according to guideline OECD 218, the amount of food necessary for the complete test duration should be added to the formulated sediment before the stabilisation period. However, in a corresponding initial main test, the emergence rate in the controls was invalid. To ensure a sufficient supply of the necessary essential ingredients for a valid emergence, a combined feeding design of plant material added to the formulated sediment before the stabilisation period and additional donations of small amounts of Tetra-Min fish food at chosen days was applied.

- Food type: 0.5 % (dry weight) of a mixture of finely ground leaves of stinging nettle (Urtica dioica) and lupine (Lupinus spec.) and Tetra-Min fish food
- Amount: The amount of fish food added was 0.125 - 0.015 mg/(day*larvae), decreasing with time. This corresponds with 25 % (day 0) and 8 – 1 % (day 2 – 15) of the maximum food amount recommended in the guideline.
- Frequency: Fish food was applied on day 0, 2, 5, 8 and 15.

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

Hardness:

- at test start: 230 – 240 mg/L as CaCO3 equivalents
- total hardness at test end: 280 – 310 mg/L as CaCO3 equivalents (controls), 260 mg/L as CaCO3 equivalents (in one representative replicate of the highest test concentration)

Test temperature:

20.5 – 20.6 °C

pH:

8.32 – 8.96

Dissolved oxygen:

72.8 - 93.5% at test start and 90.9 - 99.8% at test end (Day 29)

Ammonia:

0.5 - 0.8 mg/L NH4 (day 1), <0.1 - 7 mg/L (day 29)

Nominal and measured concentrations:

Nominal concentration: 0 (control), 46.9, 93.8, 187.5, 375, 750, and 1500 mg test item/kg dry sediment (33.5, 67.0, 134, 268, 536, and 1072 mg test item/kg wet sediment, considering a mean dry mass content of 71.5 % in the wet sediment), corresponding with 9.15, 18.3, 36.6, 73.1, 146, and 293 mg a.s./kg dry sediment (6.54, 13.1, 26.1, 52.3, 105, and 209 mg a.s./kg wet sediment)
Mean initial measured concentrations (sediment): 16.4, 34.0, 70.7, 138, 274, and 579 mg/kg dry sediment (3.20, 6.63, 13.8, 26.9, 53.4, 113 mg a.s. per kg dry sediment), representing 11.7, 24.3, 50.5, 98.6, 196 and 414 mg/kg wet sediment (2.29, 4.74, 9.85, 19.2, 38.2 and 80.7 mg a.s. per kg wet sediment), considering a mean dry mass content of 71.5 % in the wet sediment measured at test start and end after complete discharging of the overlying water.
Measured concentrations (overlaying water): < LOQ in all samples

Details on test conditions:

TEST SYSTEM
- Test container: Round glass beakers (600 mL) were used as test Vessels.
- Weight of wet sediment with and without pore water: 124 g of wet artificial sediment (corresponding with 95 g dry mass)
- Depth of sediment and overlying water: sediment: 1.5 cm, overlaying water: 6 cm high (ratio sediment:water about 1:4).
- Aeration: yes
- Aeration frequency and intensity: through a glass pipette fixed 2-3 cm above the sediment layer (at least 1 bubble /second)

EXPOSURE REGIME
- No. of organisms per container (treatment): 20 larvae
- No. of replicates per treatment group: 4
- No. of replicates per control: 6
- Feeding regime: Due to the strong adsorbing character of the test item, according to guideline OECD 218, the amount of food necessary for the complete test duration should be added to the formulated sediment before the stabilisation period. In an initial main test this procedure affected the emergence rate in the controls negatively. Therefore, and to ensure a sufficient supply of the necessary essential ingredients for a valid emergence, a combined feeding design of plant material added to the formulated sediment before the stabilisation period and additional donations of small amounts of Tetra-Min fish food at chosen days was applied. According to the guideline, plant material was used. An amount of 0.5 % (dry weight) of a mixture of finely ground leaves of stinging nettle (Urtica dioica) and lupine (Lupinus spec.) was used. Fish food was applied on day 0, 2, 5, 8 and 15. The amount added was 0.125 - 0.015 mg/(day*larvae), decreasing with time. This corresponds with 25 % (day 0) and 8 – 1 % (day 2 – 15) of the maximum food amount recommended in the guideline.

OVERLYING WATER CHARACTERISTCS
- Type of water: purified tap water

CHARACTERIZATION OF ARTIFICIAL SEDIMENT
Artificial sediment components
Sphagnum peat, air-dried, finely ground: 5 %
Kaolinite, air-dried: 20 %
Industrial quartz sand, air-dried: 75 %
- Method of preparation: The test substrate was wetted with deionised water to reach a water content of between 30% and 50%. Pulverized calcium carbonate of chemically pure quality (CaCO3) was added to adjust the pH of the final mixture of the sediment to 7.0 ± 0.5.
- Total organic carbon (%): 1.75 %

OTHER TEST CONDITIONS
- Photoperiod: 16 hours
- Light intensity: 457 – 508 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- emergence and developement time

Reference substance (positive control):

yes

Remarks:

2-chloroacetamid; However no test is required, the sensitivity was checked in a previous non-GLP spiked sediment test.

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

26.9 mg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

26.9 mg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

other: development time

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

73.1 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: development time

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

73.1 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Details on results:

The NOEC (no observed effect concentration) for emergence rate due to Amines, N-C10-C16-alkyltrimethylenedi-, reaction products with chloroacetic acid was found to be 138 mg test item per kg dry sediment (98.5 mg test item/kg wet sediment), representing 26.9 mg a.s. per kg dry sediment (19.2 mg a.s./kg wet sediment).
The NOEC (no observed effect concentration) for development time and consequently also development rate due to the test item was found to be 138 mg test item per kg dry sediment (98.5 mg test item/kg wet sediment), representing 26.9 mg a.s. per kg dry sediment (19.2 mg a.s./kg wet sediment).

Results with reference substance (positive control):

The most sensitive endpoint was emergence. The EC50 for emergence was 118 mg/kg. The EC10 was found to be 42 mg/kg. Affecting of development rate started at comparable concentrations. The EC10 values for development rate of females, males, and overall were 42 mg/kg, 48 mg/kg, and 44 mg/kg.

Reported statistics and error estimates:

For each replicate, the emergence rate was calculated as the number of emerged divided by the number of introduced individuals measured at the end of the test. In addition, the mean development rate (unit: 1/day) representing that portion of larval development, which takes place per day, was calculated. The development rate is the reciprocal of the development time, the mean time span between the introduction of larvae (day 0 of the test) and the emergence of the experimental cohort of midges. (For the calculation the age of larvae at the time of introduction was considered). Since there were no indications of statistically different sensitivities of sexes, male and female results were pooled for statistical analyses. The data were analysed using a regression model (Probit analysis assuming log-normal distribution of the values) in order to estimate the concentration that would cause x % reduction in emergence (e.g. EC10, EC50 etc.). In addition, mean values per treatment level were compared with control values to determine the lowest observed effect concentration (LOEC) and hence the no observed effect concentration (NOEC) by suitable statistical methods. The sensitivity differences between sexes were statistically judged by a Chi2-r x 2 table test. Statistical calculations were done with ToxRat® Pro 2.10, a statistical software package for ecotoxicity response analysis by ToxRat® Solutions.

Table 1: Measured concentrations of the test item. Concentration in the sediment and overlaying water [mg/kg and mg/L] and percent recovery [%] in the respective matrix of the dose introduced into the test vessel at test start, day 7, day 14 and test end. TI = Test item; DM = Dry mass.

Nominal values (per kg dry mass sediment)
Matrix	46.9 mg TI per kg DM	93.8 mg TI per kg DM	187.5 mg TI per kg DM	375 mg TI per kg DM	750 mg TI per kg DM	1500 mg TI per kg DM
test start
Sediment	16.4 mg/kg 35.0 %	34.0 mg/kg 36.2 %	70.7 mg/kg 37.7 %	137.8 mg/kg 36.7 %	273.7 mg/kg 36.5 %	579.0 mg/kg 38.6 %
Overlaying water				< LOQ		< LOQ
day 7
Sediment				220.4 mg/kg 58.8 %
day 14
Sediment				89.4  mg/kg 23.8 %
test end
Sediment	10.6 mg/kg 22.7 %			79.4 mg/kg 21.2 %		358.6 mg/kg 23.9 %
Overlaying water	< LOQ			< LOQ		< LOQ

 

Table2: Emergence at test end. Emerged midges [Ind.] and emergence rate [% of introduced larvae]. TI = Test item; DM = Dry mass; Concentrations given as initial measured values. *Significant deviation when compared with control (Fisher`s Exact Binomial Test with Bonferroni Correction, p < 0.05; one-sided).

Control	16.4 mg TI per kg DM	34.0 mg TI per kg DM	70.7 mg TI per kg DM	138 mg TI per kg DM	274 mg TI per kg DM	579 mg TI per kg DM
Emerged midges []
96	67	64	62	62	40	18
Emergence rate midges [%]
80	84	80	77	77	50 *	22 *

 

Table 3: Development time and rate. Development time [d] and rate [1/d] of midges corrected for the age of larvae introduced (1 day old).TI = Test item; DM = Dry mass. Concentrations given as initial measured values. * Significant deviation when compared with control (Williams Multiple Sequential t-test, p < 0.05; one-sided).

Control	16.4 mg TI per kg DM	34.0 mg TI per kg DM	70.7 mg TI per kg DM	138 mg TI per kg DM	274 mg TI per kg DM	579 mg TI per kg DM
Development time midges
18.4	18.7	17.7	18.5	17.8	20.4 *	23.9 *
Development rate midges
0.055	0.054	0.057	0.054	0.056	0.049 *	0.042 *

 

Table 4: Effect summary table. Effect values (mg/kg sediment) based on initial measured concentrations. TI = Test item; a.s. = active substance; C.L.= Confidence limits.

Based on dry mass sediment
Concentration	Emergence rate		Development time		Development rate
Unit	mg a.s./kg	mg TI/kg	mg a.s./kg	mg TI/kg	mg a.s./kg	mg TI/kg
EC50 (95% CL)	73.3 (53.4 - 119)	376 (274 - 611)	> 113 (n.d.)	> 579 (n.d.)	> 113 (n.d.)	> 579 (n.d.)
EC10 (95% CL)	28.1 (12.9 – 40.0)	144 (65.9 - 205)	37.8 (18.5 - 186)	194 (94.7 - 952)	37.8 (18.5 - 186)	194 (94.7 - 952)
NOEC	26.9	138	26.9	138	26.9	138
Based on fresh mass sediment
Concentration	Emergence rate		Development time		Development rate
Unit	mg a.s./kg	mg TI/kg	mg a.s./kg	mg TI/kg	mg a.s./kg	mg TI/kg
EC50 (95% CL)	52.4 (38.2 – 85.2)	269 (196 - 437)	> 80.7 (n.d.)	> 414 (n.d.)	> 80.7 (n.d.)	> 414 (n.d.)
EC10 (95% CL)	20.1 (9.19 – 28.6)	103 (47.1 - 147)	27.0 (13.2 - 133)	139 (67.7 - 680)	27.0 (13.2 - 133)	139 (67.7 - 680)
NOEC	19.2	98.5	19.2	98.5	19.2	98.5

n.d. = not determined due to mathematical reasons

 

Validity criteria fulfilled:

yes

Conclusions:

Amines, N-C10-C16-alkyltrimethylenedi-, reaction products with chloroacetic acid had an adverse effect on development and emergence of Chironomus riparius under the chosen test conditions. The relevant NOEC is 138 mg test item per kg dry sediment (98.6 mg TI/kg wet sediment), representing 26.9 mg a.s. per kg dry sediment (19.2 mg a.s./kg wet sediment) based on initial measured concentrations.

Executive summary:

The influence of Amines, N-C10-C16-alkyltrimethylenedi-, reaction products with chloroacetic acid on emergence of midges of the species Chironomus riparius was investigated. For this, the sediment-dwelling larvae (first instar) of the freshwater dipteran were placed in a sediment-water test system with defined artificial sediment. The sediment was spiked with the test item at nominal concentrations of 46.9, 93.8, 187.5, 375, 750, and 1500 mg test item per kg dry sediment, corresponding with 9.15, 18.3, 36.6, 73.1, 146, and 293 mg a.s./kg dry sediment, respectively. A single application of the test item was made. Chironomid emergence was measured at the end of the test after 28 days of incubation as the endpoint.

 

The initial measured test item concentrations in the sediment were between 35 % and 39% of nominal concentrations. The effects on the chironomids were related to the initial measured concentrations. Concentrations applied for statistical evaluation were 16.4, 34.0, 70.7, 138, 274, and 579 mg test item per kg dry sediment (3.20, 6.63, 13.8, 26.9, 53.4, 113 mg a.s. per kg dry sediment), representing 11.7, 24.3, 50.5, 98.6, 196 and 414 mg test item per kg wet sediment (2.29, 4.74, 9.85, 19.2, 38.2 and 80.7 mg a.s. per kg wet sediment), considering a mean dry mass content of 71.5 % in the wet sediment measured at test start and end after complete discharging of the overlying water.

 

An effect on emergence rate and development time or rate due to Amines, N-C10-C16-alkyltrimethylenedi-, reaction products with chloroacetic acid was detected when compared to the control midges. There was no effect on sensitivity of the sexes depending on the test item. The NOECs (no observed effect concentrations) for emergence rate and development time or rate were found to be 138 mg test item per kg dry sediment (98.6 mg TI/kg wet sediment), representing 26.9 mg a.s. per kg dry sediment (19.2 mg a.s. per kg wet sediment).

Reference 2

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-11-11 to 2014-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 225 (Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment)

Deviations:

yes

Remarks:

Accidently, 3 out of 4 replicates of the lowest treatment group (156.3 mg test item/kg dwt) were loaded with 20 instead of 10 worms. Therefore, the lowest treatment group was neglected for evaluation, due to lack of effects in the 2nd treatment group.

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen

Analytical monitoring:

yes

Details on sampling:

For the sampling time day 0, additional vessels were prepared. At the end of the test sediment and overlaying water from the main test were used.

SEDIMENT
- Concentrations: 0 (control) 156.3, 312.5, 625, 1250, 2500, and 5000 mg test item/kg dry sediment (from one vessel per treatment at test start, from one vessel of the 1250 and 5000 mg test item/kg dry sediment (at test end)

OVERLYING WATER
- Concentrations: from one vessel of the 1250 and 5000 mg test item/kg dry sediment (at the start and at the end of the test)

Vehicle:

no

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Details of spiking: test item was applied via a stock solution of the test item dissolved in deionised water. The sediment was filled in the test vessel. The quantity of stock solution required to obtain the desired concentration was added to the sediment. Subsequently, the sediment was mixed thoroughly with the applied stock solution by hand mixing. The overlaying water was filled in the vessels carefully to avoid sediment raise.
- Equilibration time: 2 days
- Controls: formulated sediment only

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Blackworm
- Strain/clone: Müller
- Source: ECT Oekotoxikologie GmbH, Flörsheim a. M., Germany. Specimens used in the test were bred in the laboratory of the Fraunhofer IME
- Breeding conditions: The breeding conditions followed Annex 5 of the OECD guideline 225. On a layer quartz sand purified tap water is used.
- Food type: a suspension of powdered TetraMin® Hauptfutter (Tetra Werke, Melle, Germany).
- Pretreatment: 10 to 14 days before the start of exposure, the worms were artificially fragmented (synchronisation). Large (adult) worms were selected for synchronisation. These worms were placed onto a glass slide in a drop of culture water, and dissected in the median body region with a scalpel. Care was taken that the posterior ends were of similar size. The posterior ends were then left to regenerate new heads in a culture vessel containing the same substrate as used in the culture and reconstituted water until the start of exposure. The test organisms were thereafter expected to be in a similar physiological state. Feeding of the synchronised worms is done once 7 d after dissection. The feeding regimen was comparable to the regular cultures, the food source the same as used in the test. The worms were held at test temperature, at 20 ± 2°C. After regenerating, intact complete worms, which were actively swimming or crawling upon a gentle mechanical stimulus, were used for the test. Injuries or autotomy in the worms were prevented, e.g. by using pipettes with fire polished edges, or stainless steel dental picks for handling these worms.

- Feeding during test: Since food was added to the sediment prior to application of the test substance, the worms were not fed additionally during the test.

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

Hardness:

290 - 310 mg/L as CaCO3

Test temperature:

18.0 – 20.2 °C

pH:

7.8 – 8.6

Dissolved oxygen:

82.2 - 94.4% oxygen saturation

Ammonia:

0.3 - 1.1 mmol/L NH4 at test start
4.1 - 7.0 mmol/L NH4 at test end

Nominal and measured concentrations:

Nominal concentration: 0 (control), 156.3, 312.5, 625, 1250, 2500, and 5000 mg test item/kg dry sediment (111, 221, 443, 886, 1771, and 3542 mg test item/kg wet sediment, considering a mean dry mass content of 70.8 % in the wet sediment), corresponding with 31.3, 62.5, 125, 250, 500, and 1000 mg a.s./kg dry sediment (22.1, 44.3, 88.6, 177, 354, and 708 mg a.s./kg wet sediment)
According to the OECD Guidance 225 the initial measured concentration should be used if the recovery in the analytic evaluation was not in the range of 80-120%. However, the initial measured value would be misleading in this special case due to the fact that the measured concentrations showed an unexpected increase from initial measured concentrations to the measured end concentrations. Therefore, a mean value was considered to express the concentration during the test duration in a conservative way. Instead of using the geometric mean which is mainly used for degradation and half-life processes (ENV/JM/MONO(2002)15) the arithmetic mean measured concentration was derived, which is also in line with the Guidance document on aquatic toxicity testing of difficult substances and mixtures (ENV/JM/MONO(2000)6). The Guidance furthermore propose to use the nominal concentration for tests with chemicals that cannot be quantified.

Arithmetic mean measured concentrations: 96, 182, 370, 714, 1465, and 2872 mg test item per kg dry sediment (18.8, 35.4, 72.1, 139, 286, 560 mg a.s. per kg dry sediment), representing 68.2, 129, 262, 506, 1038, and 2035 mg test item per kg wet sediment (13.3, 25.1, 51.1, 98.7, 202, 397 mg a.s. per kg wet sediment), considering a mean dry mass content of 70.8 % in the wet sediment.

Details on test conditions:

TEST SYSTEM
- Test container: Round glass beakers (600 mL)
- Weight of wet sediment with and without pore water: 122 g of wet artificial sediment (corresponding with 95 g dry mass).
- Depth of sediment and overlying water: The height was 1.5 cm. The overlaying water was 6 cm high (ratio sediment:water about 1:4)
- Aeration: yes
- Aeration frequency and intensity: Aeration of overlaying water was provided by pumping room air through a glass pipette fixed 2-3 cm above the sediment layer (2 - 4 bubble /second).

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 6
- Feeding regime: food was added to the sediment prior to application of the test substance, the worms were not fed additionally during the test

OVERLYING WATER CHARACTERISTCS
- Type of water: Reconstituted water according to OECD Guideline 203. At the start of the test, the pH of the test water was measured and found to be at a value of 6 to 9. The total hardness was at mean 300 mg/L as CaCO3.

CHARACTERIZATION OF ARTIFICIAL SEDIMENT
- Composition: sphagnum peat, air-dried, finely ground: 5 %, kaolinite, air-dried: 20 %, industrial quartz sand, air-dried: 75 %, in addition to the dry sediment components: powdered leaves of Stinging Nettle (Urtica sp.) 0.45 % of sediment d.w.
- Method of preparation (if artificial substrate): test substrate was wetted with deionised water to reach a water content of between 30 % and 50 %. Pulverized calcium carbonate of chemically pure quality (CaCO3) was added to adjust the pH of the final mixture of the sediment. However, since ammonia development was expected due to results of the range finding test, according to the guideline with 6.3 the pH of the sediment was kept below 7.0 but in the range of 6.0 - 6.5.
- Total organic carbon (%): 2.49

OTHER TEST CONDITIONS
- Photoperiod: 16 h/day
- Light intensity: 100 – 500 lux

EFFECT PARAMETERS MEASURED:
behavioural differences in the worms, total number of living and dead individuals per replicate (test end), differences in appearance, mortality (no reaction after a gentle mechanical stimulus and signs of decomposition or missing worms), dry weight, total biomass per replicate including adult and young worms

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Results used to determine the conditions for the definitive study: effects only at concentrations > 500 mg test item/kg dry sediment, EC50 in the range of 4000 – 6000 mg test item/kg dry sediment.



For the preparation of the stock solutions for mineral medium the following reagents (analytical grade) were used:
( a ) CaCl2 x 2 H2O 117.6 g/L
( b ) MgSO4 x 7 H2O 49.3 g/L
( c ) NaHCO3 25.9 g/L
( d ) KCl 2.3 g/L

The reconstituted water applied in the test contained 2.5 mL/L of the mineral stock solution (a) – (d).

Reference substance (positive control):

yes

Remarks:

pentachlorophenol (PCP); A test with the reference substance is regularly performed at the test facility once a year.

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

26.7 mg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

(yield)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

26.7 mg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

other: living worms

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

98.1 mg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

218.1 mg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

other: survival rate

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

35.4 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: living worms

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

139 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

35.4 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

(yield)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

286 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: survival rate

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

488 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: survival rate

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

60.9 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

(yield)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

60.9 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: living worms

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

244 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

The NOEC (no observed effect concentration) for total number of living worms due to Amines, N-C10-C16-alkyltrimethylenedi-, reaction products with chloroacetic acid was found to be 182 mg test item per kg dry sediment (129 mg/kg wet sediment), representing 35.4 mg a.s. per kg dry sediment (25.1 mg/kg wet sediment).
The NOEC (no observed effect concentration) for biomass was found to be 714 mg test item per kg dry sediment (506 mg/kg wet sediment), representing 139 mg a.s. per kg dry sediment (98.7 mg/kg wet sediment).
The NOEC (no observed effect concentration) for the survival rate was found to be 1465 mg test item per kg dry sediment (1038 mg/kg wet sediment), representing 286 mg a.s. per kg dry sediment (202 mg/kg wet sediment).
The NOEC (no observed effect concentration) for reproduction was found to be 182 mg test item per kg dry sediment (129 mg/kg wet sediment), representing 35.4 mg a.s. per kg dry sediment (25.1 mg/kg wet sediment).
The test item had adverse effect on number of total living worms, biomass, survival rate, and reproduction of Lumbriculus variegatus under the chosen test conditions. The relevant NOEC is 182 mg test item per kg dry sediment (129 mg/kg wet sediment), representing 35.4 mg a.s. per kg dry sediment (25.1 mg/kg wet sediment) based on arithmetic mean measured concentrations.

According to the OECD Guidance 225 the initial measured concentration should be used if the recovery in the analytic evaluation was not in the range of 80-120%. However, the initial measured value would be misleading in this special case due to the fact that the measured concentrations showed an unexpected increase from initial measured concentrations to the measured end concentrations. Therefore, a mean value was considered to express the concentration during the test duration in a conservative way. Instead of using the geometric mean which is mainly used for degradation and half-life processes (ENV/JM/MONO(2002)15) the arithmetic mean measured concentration was derived, which is also in line with the Guidance document on aquatic toxicity testing of difficult substances and mixtures (ENV/JM/MONO(2000)6). The Guidance furthermore propose to use the nominal concentration for tests with chemicals that cannot be quantified.

Results with reference substance (positive control):

The sensitivity of the test organisms was checked by using PCP as reference substance in a non-GLP 96 h acute non-GLP reference test, not older than 12 month.
Immobilization after 96 h:
Control: 0 %
31.3 µg PCP/L: 0% (related to control: 0 %)
62.5 µg PCP/L: 0% (related to control: 0 %)
125 µg PCP/L: 0% (related to control: 0 %)
250 µg PCP/L: 5% (related to control: 5 %)
500 µg PCP/L: 100% (related to control: 100 %)
96 h EC50 value:
Immobilization: 325 µg/L (95% CL: n.d. – n.d. µg/L)

Reported statistics and error estimates:

The data were analysed using a regression model (Probit analysis assuming log-normal distribution of the values) in order to estimate the concentration that would cause x % reduction in biomass and total number of worms (e.g. EC10, EC50 etc.). In addition, mean values per treatment level were compared with control values to determine the lowest observed effect concentration (LOEC) and hence the no observed effect concentration (NOEC) by suitable statistical methods. Statistical calculations were done with ToxRat® Pro 2.10, a statistical software package for ecotoxicity response analysis by ToxRat® Solutions.

Table 1: Measured concentrations of test item. Concentration in the sediment and overlaying water [mg/kg and mg/L] and percent recovery [%] in the respective matrix of the dose introduced into the test vessel at test start and test end. TI = Test item; DM = Dry mass.* Concentrations were calculated on the basis of the mean measured recovery rate of treatment 4 and 6 for arithmetic mean calculation (seeTable2).

Nominal values (per kg dry mass sediment)
Matrix	156.3 mg TI per kg DM	312.5 mg TI per kg DM	625 mg TI per kg DM	1250 mg TI per kg DM	2500 mg TI per kg DM	5000 mg TI per kg DM
test start (measured values and percent recovery)
Sediment	79.2 mg TI per kg DM 50.7 %	137 mg TI per kg DM 43.7 %	286 mg TI per kg DM 45.8 %	502 mg TI per kg DM 40.2 %	1117 mg TI per kg DM 44.7 %	2199 mg TI per kg DM 44.0 %
Overlaying water				0.46 mg/L 0.10 %		3.63 mg/L 0.21 %
test end (measured values and percent recovery)
Sediment	113 mg TI per kg DM* 72.5 %	227 mg TI per kg DM* 72.5 %	453 mg TI per kg DM* 72.5 %	926 mg TI per kg DM 74.1 %	1812 mg TI per kg DM* 72.5 %	3545 mg TI per kg DM 70.9 %
Overlaying water				< LOQ		< LOQ

 

Table 2: Arithmetic mean measured concentrations of test item. Concentration in the sediment [mg/kg] and percent recovery [%] of the dose introduced into the test vessel. TI = Test item; a.s. = active substance; DM = Dry mass.

Nominal values (per kg dry mass sediment)
Matrix	156.3 mg TI per kg DM 30.5 mg a.s. per kg DM)	312.5 mg TI per kg DM (60.9 mg a.s. per kg DM)	625 mg TI per kg DM (122 mg a.s. per kg DM)	1250 mg TI per kg DM (244 mg a.s. per kg DM)	2500 mg TI per kg DM (488 mg a.s. per kg DM)	5000 mg TI per kg DM (975 mg a.s. per kg DM)
Sediment	96.2 mg TI per kg DM (18.8 mg a.s. per kg DM) 61.6 %	182 mg TI per kg DM (35.4 mg a.s. per kg DM) 58.1 %	370 mg TI per kg DM (72.1 mg a.s. per kg DM) 59.1 %	714 mg TI per kg DM (139 mg a.s. per kg DM) 57.1 %	1465 mg TI per kg DM (286 mg a.s. per kg DM) 58.6 %	2872 mg TI per kg DM (560 mg a.s. per kg DM) 57.4 %

 

Table 3: Mean number of living worms after 28 days[Individuals]. TI = Test item; a.s. = active substance; DM = Dry mass; FM = Fresh mass; Concentrations given as arithmetic mean measured values. *Significant deviation when compared with control (Williams Multiple Sequential t-test, p < 0.05; one-sided). Number of specimens introduced at test start: 10 per replicate.

	Control	182 mg TI per kg DM (35.4 mg a.s. per kg DM)	370 mg TI per kg DM (72.1 mg a.s. per kg DM)	714 mg TI per kg DM (139 mg a.s. per kg DM)	1465 mg TI per kg DM (286 mg a.s. per kg DM)	2872 mg TI per kg DM (560 mg a.s. per kg DM)
Living worms [Ind]	18.8	19.8	14.0*	13.0*	13.3*	7.5*

 

 

Table 4: Reproduction rate after 28 days. Mean of all replicates [Yield]. Increase of worm numbers (yield) based on number of specimens introduced (10 per replicate); TI = Test item; a.s. = active substance; DM = Dry mass; FM = Fresh mass; Concentrations given as arithmetic mean measured values. *Significant deviation when compared with control (Williams Multiple Sequential t-test, p < 0.05; one-sided, p < 0.05; one-sided).

	Control	182 mg TI per kg DM (35.4 mg a.s. per kg DM)	370 mg TI per kg DM (72.1 mg a.s. per kg DM)	714 mg TI per kg DM (139 mg a.s. per kg DM)	1465 mg TI per kg DM (286 mg a.s. per kg DM)	2872 mg TI per kg DM (560 mg a.s. per kg DM)
Reproduction [Yield]	8.83	9.75	4.00*	3.00*	3.25*	-2.50*

 

Table 5: Mortality after 28 days. Mean of all replicates [%]. Decrease of worm numbers based on number of specimens introduced (10 per replicate); TI = Test item; a.s. = active substance; DM = Dry mass; FM = Fresh mass; Concentrations given as arithmetic mean measured values. *Significant deviation when compared with control (Fisher`s Exact Binomial Test with Bonferroni Correction, p < 0.05; one-sided).

	Control	182 mg TI per kg DM (35.4 mg a.s. per kg DM)	370 mg TI per kg DM (72.1 mg a.s. per kg DM)	714 mg TI per kg DM (139 mg a.s. per kg DM)	1465 mg TI per kg DM (286 mg a.s. per kg DM)	2872 mg TI per kg DM (560 mg a.s. per kg DM)
Mortality [%]	0	0	0	0	0	28*

 

Table 6: Biomass. Biomass on a per vessel basis [g].TI = Test item; a.s. = active substance; DM = Dry mass; FM = Fresh mass; Concentrations given as arithmetic mean measured values. * Significant deviation when compared with control (Welch-t test for Inhomogeneous Variances, p < 0.05; one-sided).

	Control	182 mg TI per kg DM (35.4 mg a.s. per kg DM)	370 mg TI per kg DM (72.1 mg a.s. per kg DM)	714 mg TI per kg DM (139 mg a.s. per kg DM)	1465 mg TI per kg DM (286 mg a.s. per kg DM)	2872 mg TI per kg DM (560 mg a.s. per kg DM)
Biomass [g DM]	0.030	0.035	0.021	0.025	0.018*	0.006*

 

Table 7: Effect summary table. Effect values (mg/kg sediment) based on concentrations calculated from arithmetic mean measured concentrations. TI = Test item; a.s. = active substance; C.L.= Confidence limits.

Based on dry mass sediment
Concentration	No. of living worms		Reproduction		Mortality		Biomass
Unit	mg a.s./kg	mg TI/kg	mg a.s./kg	mg TI/kg	mg a.s./kg	mg TI/kg	mg a.s./kg	mg TI/kg
EC50 (95% CL)	440 (n.d.)	2257 (n.d.)	103 (n.d.)	526 (n.d.)	788 (n.d.)	4041 (n.d.)	295 (n.d.)	1512 (n.d.)
EC10 (95% CL)	41.1 (n.d.)	211 (n.d.)	23.4 (n.d.)	120 (n.d.)	404 (n.d.)	2072 (n.d.)	67.5 (n.d.)	346 (n.d.)
NOEC	35.4	182	35.4	182	286	1465	139	714
Based on fresh mass sediment
Concentration	No. of living worms		Reproduction		Mortality		Biomass
Unit	mg a.s./kg	mg TI/kg	mg a.s./kg	mg TI/kg	mg a.s./kg	mg TI/kg	mg a.s./kg	mg TI/kg
EC50 (95% CL)	312 (n.d.)	1599 (n.d.)	72.7 (n.d.)	373 (n.d.)	558 (n.d.)	2863 (n.d.)	209 (n.d.)	1071 (n.d.)
EC10 (95% CL)	29.1 (n.d.)	149 (n.d.)	16.6 (n.d.)	85.0 (n.d.)	286 (n.d.)	1468 (n.d.)	47.8 (n.d.)	245 (n.d.)
NOEC	25.1	129	25.1	129	202	1038	98.7	506

n.d. = not determined due to mathematical reasons

Validity criteria fulfilled:

yes

Conclusions:

Amines, N-C10-C16-alkyltrimethylenedi-, reaction products with chloroacetic acid had adverse effects on the number of total living worms, biomass, survival rate, and reproduction of Lumbriculus variegatus in a toxicity test using spiked sediment according to OECD 225. The relevant NOEC is 182 mg test item per kg dry sediment (129 mg TI/kg wet sediment), representing 35.4 mg a.s. per kg dry sediment (25.1 mg a.s./kg wet sediment) based on arithmetic mean measured concentrations.

Executive summary:

The influence of Amines, N-C10-C16-alkyltrimethylenedi-, reaction products with chloroacetic acid on development of the endobenthic oligochaete Lumbriculus variegatus was investigated. For this, worms of similar physiological state were placed in a sediment-water test system with defined artificial sediment. The sediment was spiked with the test item at nominal concentrations of 156.3, 312.5, 625, 1250, 2500 and 5000 mg test item per kg dry sediment, corresponding to 30.5, 60.9, 122, 244, 488, and 975mg a.s./kg dry sediment, respectively. A single application of the test item was made. Mortality, reproduction rate and biomass were measured at the end of the test after 28 days of incubation as the endpoint.

The initial measured test item concentrations in the sediment were between 40 % and 51 % of nominal concentrations of the lead compounds N-C12 Gly and N’-C12 Gly. The test item concentrations measured in two representative treatment levels (1250 and 5000 mg test item per kg dry sediment) at test end were 74 and 71 %, respectively. Since sensitivity of test specimens is assumed to maintain constant during the test, effects on the worms were related to the arithmetic mean measured concentrations of test start and test end. Concentrations applied for statistical evaluation were 96, 182, 370, 714, 1465, and 2872 mg TEST ITEM per kg dry sediment (18.8, 35.4, 72.1, 139, 286, 560 mg a.s. per kg dry sediment), representing 68.2, 129, 262, 506, 1038, and 2035 mg TEST ITEM per kg wet sediment (13.3, 25.1, 51.1, 98.7, 202, 397 mg a.s. per kg wet sediment), considering a mean dry mass content of 70.8 % in the wet sediment.

An effect on total number of living worms and biomass due to TEST ITEM was detected when compared to the control worms. The NOECs for both endpoints were found to be 182 mg TEST ITEM per kg dry sediment (129 mg TI/kg wet sediment), representing 35.4 mg a.s. per kg dry sediment (25.1 mg a.s. /kg wet sediment) for total number of living worms and 714 mg TEST ITEM per kg dry sediment (506 mg TI/kg wet sediment), representing 139 mg a.s. per kg dry sediment (98.7 mg a.s./kg wet sediment) for biomass, respectively.

Also survival rate and reproduction (yield) was affected by the test item. The NOECs for both endpoints were found to be 1465 mg TEST ITEM per kg dry sediment (1038 mg TI/kg wet sediment), representing 286 mg a.s. per kg dry sediment (202 mg a.s./kg wet sediment) for survival rate and 182 mg TEST ITEM per kg dry sediment (129 mg TI/kg wet sediment), representing 35.4 mg a.s. per kg dry sediment (25.1 mg a.s./kg wet sediment) for reproduction (yield), respectively.

Reference 3

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2015-02-05 to 2015-03-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA/600/R-94/064

Principles of method if other than guideline:

The objective of the study is the assessment of the effects of prolonged exposure of the amphipod Hyalella azteca to sediment-associated chemicals. The exposure scenario used in this guideline is spiking of sediment with the test substance. Using spiked sediment is intended to simulate a sediment contaminated with the test compound. This exposure system involves spiking sediment of a sediment-water test system. The test method is aimed to determine effects of a test substance on the growth (biomass), and survival of the organisms. Ten young Hyalella are placed in a sediment-water test system with defined artificial sediment. The sediment is spiked with the test item in different concentrations. A single application of the test item will be made. It is a static test without water renewal, except for replacement of evaporative losses and water chemistry measurements.
The test principles followed Borgmann, U. (2003) , USEPA (EPA/600/R-99/064) and Borgmann, U. and Norwood, W.P. (1997) . The measured biological parameters were the total number of surviving Hyalella and the biomass (dry weight) measured at the end of the exposure.
Using appropriate statistical methods it was analyzed, whether there was a statistical significant difference in survival and biomass between the treatment and the control.

GLP compliance:

yes (incl. QA statement)

Remarks:

2011-02-07

Analytical monitoring:

yes

Details on sampling:

SEDIMENT
- Concentrations: The sediment was spiked with the test item at nominal concentrations of 83, 208, 520, 1300, 3250 mg test item per kg dry sediment, corresponding with 16.2, 40.6, 101, 254 and 634 mg a.s. per kg dry sediment, respectively.
- Sampling interval: Survival and biomass was measured at the end of the test after 28 days as the endpoint.

Vehicle:

no

Details on sediment and application:

CHARACTERIZATION OF (ARTIFICIAL) SEDIMENT
Artificial sediment components:
a) Sphagnum peat, air-dried, finely ground (≤ 1 mm) 5 %
b) Kaolinite, air-dried 20 %
c) Industrial quartz sand, air-dried 75 %
d) Powdered leaves of lucerne, at 0.5 % of sediment d.w., in addition to the dry sediment components.

The test substrate was wetted with deionised water to reach a water content of between 30 % and 50 %. Pulverized calcium carbonate of chemically pure quality (CaCO3) was added to adjust the pH of the final mixture of the sediment to 7.0 ± 0.5. Organic carbon content of the final mixture was 1,8 %, which was in the demanded range of 2 % ± 0.5 %.

PREPARATION OF SPIKED SEDIMENT
The liquid test item was applied via stock solution of the test item dissolved in “Hyalella”-media. The quantity of stock solution required to obtain the desired concentration was added to the sediment. The respective portion of sediment (adjusted to 20 – 40 % water content to avoid waterlogging during distribution) was filled in the test vessel.
Subsequently, the sediment was mixed thoroughly with the applied stock solution by hand mixing. Subsequently, the respective subsample of the spiked sediment is filled in the test vessels. The overlaying water was filled in the vessels carefully to avoid sediment raise. To reach equilibrium of test item distribution within the matrices, the assays were aged for two days before test start (introduction of the Hyalella).

Test organisms (species):

Hyalella azteca

Details on test organisms:

TEST ORGANISM
Test organism was Hyalella azteca. Origin of the Hyalella is Freds Haustierzoo (Berliner Straße 37, 51063 Köln). Specimens used in the test were bred in the laboratory at Fraunhofer IME.

Breeding conditions:
Hyalella were hold in ca. 2 L of “Hyalella”-media-water [3] [Table 1Table 1] with a small piece ~20 cm2 of a cotton gauze (Lohmann&Rauscher) as substrate in the vessels. The temperature of the “Hyalella”-media was 25 ± 2°C, the photoperiod 16/8h light-dark cycle, the light intensity 500-1000 Lux, and pH 6 to 8.
Hyalella were fed with grounded TetraMin® (Tetra Werke, Melle, Germany) twice a week ad libitum (small spoon: ~0.06 g). Hyalella were sieved weekly with two different net widths. Adults stayed in the net above (mesh width: 560 µm), juvenils stayed in the smaller net below (mesh width: 180 µm).
Juveniles were 10-16 days old at test start.

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

Hardness:

Total hardness at test start was 3.5 nmol/L – 3.9 nmol/L and at test end 2.3 nmol/L –2.7 nmol/L

Test temperature:

22.3 – 23.4 °C

pH:

8.1 – 8.6

Dissolved oxygen:

Oxygen saturation was 60 % – 102%

Salinity:

Nitrite was 0.01 mg/L – 3.43 mg/L, and nitrate 2 mg/L – 9 mg/L

Ammonia:

0.1 mg/L – 7.9 mg/L

Nominal and measured concentrations:

The nominal concentration in the test containers with test item was 83.2, 208, 520, 1300, and 3250 mg test item per kg dry sediment (16.2, 40.6, 101, 254, and 634 mg test item a.s./kg dry sediment).
Concentration applied for statistical evaluation were 26.3, 71.3, 197, 439, and 1102 mg test item per kg dry sediment (5.13, 13.9, 38.4, 85.6, 215 mg a.s. per kg dry sediment), representing 18.6, 50.5, 140, 311, and 781 mg test item per kg wet sediment (3.63, 9.85, 27.2, 60.6, 152 mg a.s. per kg wet sediment), considering a mean dry mass content of 70.8 % in the wet sediment measured at test start and end after complete discharging of the overlying water.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Glass wide bottles (250 mL, 6 cm ∅) were used as test vessels.
- Sediment volume: The test vessels were filled up with an amount of artificial sediment, resulting in a depth of the sediment layer of ~1.3 cm (~40 ml).
- Overlying water volume: The overlaying water had a ratio from sediment: water about 1:4, the water volume is ~160 ml.
- Aeration: yes
- Aeration frequency and intensity: Aeration of overlaying water were provided through an cannula driven through screw cap of the glass and fixed 1-2 cm in the water phase.
- Replacement of evaporated test water, if any: yes

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 6
- No. of replicates per control: 6
- Feeding regime: Since food was added to the sediment before test start (right before application of the test substance), the Hyalella were not fed additionally during the test.
- Type and preparation of food: Powdered leaves of lucerne, at 0.5 % of sediment d.w., in addition to the dry sediment components.

OVERLYING WATER CHARACTERISTCS
“Hyalella”-Media is used as test water above the sediment.

Composition of the artificial “Hyalella”- media according to Borgmann, 1996
Salt Concentration [mM]
CaCl2 1
NaBr 0,01
KCl 0.05
NaHCO3 1
MgSO4 0.25

CHARACTERIZATION OF (ARTIFICIAL; delete if not applicable) SEDIMENT
Artificial sediment components:
a) Sphagnum peat, air-dried, finely ground (≤ 1 mm) 5 %
b) Kaolinite, air-dried 20 %
c) Industrial quartz sand, air-dried 75 %
d) Powdered leaves of lucerne, at 0.5 % of sediment d.w., in addition to the dry sediment components.

The test substrate was wetted with deionised water to reach a water content of between 30 % and 50 %. Pulverized calcium carbonate of chemically pure quality (CaCO3) was added to adjust the pH of the final mixture of the sediment to 7.0 ± 0.5. Organic carbon content of the final mixture was 1,8 %, which was in the demanded range of 2 % ± 0.5 %.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours
- Light intensity: 500-1000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Survival
- Biomass

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

38.4 mg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

other: survival

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

38.4 mg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

other: biomass (per vessel)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

101 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: survival

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

101 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: biomass (per vessel)

Details on results:

The NOECs, EC10 and EC50 values for survival of Hyalella and biomass are presented in tables below..

The NOEC (no observed effect concentration) for survival was found to be 197 mg test item per kg dry sediment (140 mg test item/ kg wet sediment), representing 38.4 mg a.s. per kg dry sediment (27.2 mg a.s./ kg wet sediment).
The NOEC (no observed effect concentration) for biomass per vessel was found to be 197 mg test item per kg dry sediment (140 mg test item/ kg wet sediment), representing 38.4 mg a.s. kg dry sediment (27.2 mg a.s./ kg wet sediment).

The test item had adverse effect on survival and biomass of Hyalella atzeca under the chosen conditions. The relevant NOEC is 197 mg test item per kg dry sediment (140 mg/kg wet sediment), representing 38.4 mg a.s. kg dry sediment (27.2 mg/kg wet sediment).

Results with reference substance (positive control):

none

Reported statistics and error estimates:

For each concentration, the survival of Hyalella, percent mortality (decrease based on the number of Hyalella introduced at test start), and the biomass of all Hyalella were determined.
The main response variables of the test to be evaluated statistically are the biomass and the survival of Hyalella per replicate.
The data were analysed using a regression model (Probit analysis) in order to estimate the concentration that would cause x % reduction in biomass and suvival of Hyalella (e.g. EC10, EC50 etc.). In addition, mean values per treatment level were compared with control values to determine the lowest observed effect concentration (LOEC) and hence the no observed effect concentration (NOEC) by suitable statistical methods. Statistical calculations were done with ToxRat® Pro 2.10, a statistical software package for ecotoxicity response analysis by ToxRat® Solutions.

Table A7.4.3.5.1-2: Living Hyalella after 28 days.Sum of all replicates [Individuals].TI = Test item; DM = Dry mass; a.s. = active substance. Concentrations given as initial measured values. *Significant deviation when compared to control (Fisher’s Exact Binomial Test with Bonferroni Correction, α = 0.05, one-sided greater)

	Control	26.3 mg TI per kg DM (5.13 mg a.s. per kg DM)	71.3 mg TI per kg DM^1(13.9 mg a.s. per kg DM)	197 mg TI per kg DM (38.4 mg a.s. per kg DM)	439 mg TI per kg DM (85.6 mg a.s. per kg DM)	1102 mg TI per kg DM (215 mg a.s. per kg DM)
LivingHyalella [Ind]	57^2	58	52	60	44*	22*

¹concentration calculated based on mean percentual relative recovery of all measured concentration

²in one replicate were found 11Hyalella azteca

 

Table A7.4.3.5.1-3:Mortality after 28 days.Mean of all replicates[%].Decrease ofHyalellanumbers based on number of specimens introduced (10 per replicate, in one control replicate were found 11Hyalella); TI = Test item; DM = Dry mass; a.s. = active substance. Concentrations given as initial measured values. *Significant deviation when compared to control (Fisher’s Exact Binomial Test with Bonferroni Correction, α = 0.05, one-sided greater)

	Control	26.3 mg TI per kg DM (5.13 mg a.s. per kg DM)	71.3 mg TI per kg DM^1(13.9 mg a.s. per kg DM)	197 mg TI per kg DM (38.4 mg a.s. per kg DM)	439 mg TI per kg DM (85.6 mg a.s. per kg DM)	1102 mg TI per kg DM (215 mg a.s. per kg DM)
Mortality [%]	6.6	3.3	13.3	0.00	26.7*	63.3*

¹concentration calculated based on mean percentual relative recovery of all measured concentration

 

Table A7.4.3.5.1-4:Biomass per vessel. Biomass on a per vessel basis.TI = Test item; DM = Dry mass; a.s. = active substance. Concentrations given as initial measured values. * Significant deviation when compared with control (Williams Multiple Sequential t-test, α = 0.05, one-sided smaller)

	Control	26.3 mg TI per kg DM (5.13 mg a.s. per kg DM)	71.3 mg TI per kg DM^1(13.9 mg a.s. per kg DM)	197 mg TI per kg DM (38.4 mg a.s. per kg DM)	439 mg TI per kg DM (85.6 mg a.s. per kg DM)	1102 mg TI per kg DM (215 mg a.s. per kg DM)
Biomass [mg DM]	2.51	2.83	2.56	2.05	1.38*	0.51*

¹concentration calculated based on mean percentual relative recovery of all measured concentration

 

Table A7.4.3.5.1-5:Biomass perHyalella. Biomass calculated for oneHyalella.TI = Test item; DM = Dry mass; a.s. = active substance. Concentrations given as initial measured values. * Significant deviation when compared with control (Williams Multiple Sequential t-test, α = 0.05, one-sided smaller)

	Control	26.3 mg TI per kg DM (5.13 mg a.s. per kg DM)	71.3 mg TI per kg DM^1(13.9 mg a.s. per kg DM)	197 mg TI per kg DM (38.4 mg a.s. per kg DM)	439 mg TI per kg DM (85.6 mg a.s. per kg DM)	1102 mg TI per kg DM (215 mg a.s. per kg DM)
Biomass [mg DM]	0.26	0.28	0.29	0.20	0.19*	0.15*

¹concentration calculated based on mean percentual relative recovery of all measured concentration

Table A7.4.3.5.1-6:Effect summary table.Effect values based on concentrations calculated from initial measured concentrations. NOEC = No observed effect concentration; EC = Effect concentration a.s. = active substance; C.L. = Confidence limits; n.d. = not determined due to mathematical reasons

Based on dry mass sediment
Concentration	Survival		Biomass (per vessel)
Unit	mg a.s./kg	mg TI/kg	mg a.s./kg	mg TI/kg
EC50	192	984	95.7	491
(95% CL lower	(n.d.)	(n.d.)	88.0	452
(95% CL upper)	(n.d.)	(n.d.)	104	535
EC10	39.7	204	27.5	141
(95% CL lower	(n.d.)	(n.d.)	22.5	115
(95% CL upper)	(n.d.)	(n.d.)	32.3	166
NOEC	38.4	197	38.4	197
Based on fresh mass sediment
Concentration	Survival		Biomass (per vessel)
Unit	mg a.s./kg	mg TI/kg	mg a.s./kg	mg TI/kg
EC50	136	697	67.8	347.8
(95% CL lower	(n.d.)	(n.d.)	62.4	319.8
(95% CL upper)	(n.d.)	(n.d.)	73.9	379.0
EC10	28.1	144	19.5	100
(95% CL lower	(n.d.)	(n.d.)	15.9	81.6
(95% CL upper)	(n.d.)	(n.d.)	22.9	117.5
NOEC	27.2	140	27.2	140

 

Validity criteria fulfilled:

yes

Remarks:

The Hyalella azteca, chronic toxicity test with sediment fulfills the validity criteria: The mortality of control animals < 20% at test end; The oxygen concentration in the overlaying water > 30% of air saturation; The pH of the overlaying water was 6-9

Conclusions:

Amines, N-C10-C16-alkyltrimethylenedi-, reaction products with chloroacetic acid had adverse effect on survival and biomass of Hyalella azteca under the chosen test conditions. The NOEC is 197 mg test item per kg dry sediment (140 mg test item/kg wet sediment) representing 38.4 mg a.s. per kg dry sediment (27.2 mg a.s./kg wet sediment) based on mean initial measured concentrations.

Executive summary:

The influence of Amines, N-C10-C16-alkyltrimethylenedi-, reaction products with chloroacetic acid on development of the amphipod Hyalella azteca to sediment-associated chemicals was investigated. For this, young Hyalella were placed in a sediment-water test system with defined artificial sediment. The sediment was spiked with the test item at nominal concentrations of 83, 208, 520, 1300, 3250 mg test item per kg dry sediment, corresponding with 16.2, 40.6, 101, 254 and 634 mg a.s. per kg dry sediment, respectively. A single application of the test item was made. Survival and biomass was measured at the end of the test after 28 days as the endpoint. The test principles are based on Borgmann, USEPA and Borgmann and Norwood.

 The initial measured test item concentrations in the sediment were between 32 % and 38 % of nominal concentrations at test start. The measured test item concentration in the sediment were between 14 % and 17 % of nominal concentration at test end. Effects on the Hyalella were related to the initial measured concentrations. Concentration applied for statistical evaluation were 26.3, 71.3, 197, 439, and 1102 mg test item per kg dry sediment (5.13, 13.9, 38.4, 85.6, 215 mg a.s. per kg dry sediment), representing 18.6, 50.5, 140, 311, and 781 mg test item per kg wet sediment (3.63, 9.85, 27.2, 60.6, 152 mg a.s. per kg wet sediment), considering a mean dry mass content of 70.8 % in the wet sediment.

Since the second concentration (208 mg test item per kg, nominal concentration) were not analyzed for the initial concentration, the value was calculated based on mean percentual relative recovery of all measured concentration. 

 An effect on survival and biomass due to test item was detected when compared to the control Hyalella. The NOECs (no observed effect concentrations) for both endpoints were found to be 197 mg test item per kg dry sediment (140 mg test item /kg wet sediment), corresponding with 38.4 mg a.s. per kg dry sediment (27.2 mg a.s./kg wet sediment) based on initial measured concentration.

Description of key information

NOEC (28 d) = 26.7 mg a.i./kg sediment dw (initial measured, Lumbriculus variegatus, OECD guideline 225)

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

26.7 mg/kg sediment dw

Additional information

Three long-term studies testing the chronic toxicity of Amines, N-C10-C16-alkyltrimethylenedi-, reaction products with chloroacetic acid to sediment dwelling organisms are available. Comparable no effect concentrations (NOEC) were determined in all three studies.

The lowest NOEC was documented in the key study following OECD 225. The effects on the number of total living worms, biomass, survival rate, and reproduction of Lumbriculus variegatus was investigated. The sediment was spiked with the test item at nominal concentrations of 156.3, 312.5, 625, 1250, 2500 and 5000 mg/kg dw, corresponding to 30.5, 60.9, 122, 244, 488, and 975mg a.i./kg dw, respectively. A single application of the test item was made. Mortality, reproduction rate and biomass were measured at the end of the test after 28 days of incubation as the endpoint. The initial measured test item concentrations in the sediment were between 40 % and 51 % of nominal concentrations of the lead compounds. The lowest effect level of 26.7 mg a.i./kg dw (initial measured) was determined for the reproduction rate of L. variegatus after 28 days of exposure. An effect on biomass was detected when compared to the control worms. The NOEC was found to be 98.1 mg a.i./kg dw for biomass (initial measured), respectively. The test item also affected the survival rate of L. variegatus. The NOECs was found to be 218 mg a.i./kg (initial measured).

A second study following OECD 218 was conducted with Chironomus riparius as test organism. The sediment was spiked with the test item at nominal concentrations of 46.9, 93.8, 187.5, 375, 750, and 1500 mg test item per kg dry sediment, corresponding with 9.15, 18.3, 36.6, 73.1, 146, and 293 mg a.i./kg dw, respectively. A single application of the test item was made. Chironomid emergence was measured at the end of the test after 28 days of incubation as the endpoint. The initial measured test item concentrations in the sediment were between 35 % and 39% of nominal concentrations. Based on the emergence of Chironomids after 28 days, a NOEC of 26.9 mg a.i./kg dw (initial measured) was determined and supports the results of the key study.

The effect of the test item on survival and biomass of Hyalella azteca was investigated in a third study conducted according to EPA/600/R-94/064. An effect on survival and biomass due to the test item was detected when compared to the control Hyalella. The NOECs for both endpoints were found to be 197 mg test item/kg dw, corresponding with 38.4 mg a.i./kg dw based on initial measured concentration.