Tributylmethylammonium chloride

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.0625, 0.125, 0.25, 0.5 and 1 mg/L
- Sampling method: Test sample of pre and post renewal purposes was collected from the test vessel at three different layers form mid, top and lower layers.
- Sample storage conditions before analysis: All test concentrations whcih were collected to analysis purposes, has been analysed immediately.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test chemical of 10 g was weighed and dissolved in the medium, for homogenisity the soluiton was kept on stirring
- Controls: M7 medium itself was used as control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea
- Strain/clone: STRAUS
- Justification for species other than prescribed by test guideline: Test organism was as per test guideline
- Age at study initiation (mean and range, SD): <24 hours, which were not from the first brood pouch
- Method of breeding: in house breeding, parent daphinds were maintained same at the test conditions in M7 medium, and fresh water algae was privided as feed
- Source: MicroBio tests Kleimoer 15B-9030 MARIAKERKE (GENT) BELGIUM
- Age of parental stock (mean and range, SD): > 3 weeks
- Feeding during test : yes provided
- Food type: Fresh water algae P subcapitata
- Amount: 0.1-0.2 mg/carbon/daphnids
- Frequency: Weekly thrice at the time of renewal

ACCLIMATION
- Acclimation period: Parent daphnids were maintined in test conditions whose age was > 3 weeks for minimum of 48 hours
- Acclimation conditions (same as test or not): same as test conditions
- Type and amount of food: Selenestrum capricornutum and amount
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): no mortality was observed

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

After exposure duration of 21 days all surviving parent daphnids were measured for their lengths

Hardness:

225

Test temperature:

19 ± 0.5°C

pH:

Control pH at Day 0 -7.5 and at Day 21 - 7.9

Dissolved oxygen:

Control DO - day 0- 7.9 and day 21- 8.1

Conductivity:

0.21 µS/cm

Nominal and measured concentrations:

Nominal Concentrations: 0.0625, 0.125, 0.25, 0.5 and 1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL beaker
- Type (delete if not applicable): Loosley closed to avoid crossover and contamination
- Material, size, headspace, fill volume: Glass beaker, 50 ml
- Aeration: No aeration
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): every alternate days thrice weekly
- No. of organisms per vessel: 1 test organism /vessel
- No. of vessels per concentration (replicates): 10 vessels per concentration
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 10 test organisms/ concentration

TEST MEDIUM / WATER PARAMETERS
- Conductivity: 0.21 µS/cm
- Culture medium different from test medium: M7 medium was used in the expermental phase and also in test organism maintenance
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: pH not adjusted
- Photoperiod: 16 hours light and 8 hours dark
- Light intensity: 1221.75 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: base on the short term toxicity concentrations for the main study was selected,

Reference substance (positive control):

not required

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

0.47 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95 % CI 0.4263- 5190

Details on results:

- Behavioural abnormalities:
- Observations on body length: length of parent daphnids after 21 days was 0.36, 0.33, 0.33, 0.32, 0.29, 0.28 cm in control, 0.0625, 0.125, 0.25, 0.5 and 1 mg/L respectively, and offsprings size was 0.08, 0.08, 0.08, 0.05, 0.05, 0.05 cm in control, 0.0625, 0.125, 0.25, 0.5 and 1 mg/L respectively
- Mortality of control: None
- Other adverse effects control: None
- Immobilisation of control: None
- Abnormal responses: none

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The NOEC was calculated using Student T test and for reproductive capacity. Probit regression analysis was used to calculate median lethal concentration with the help of statistical software Stat Plus.

Nominal Concentration:Control

Day	0	1	2	3	4	5	6	7			8	9	10	11	12	13	14	15	16	17		18	19	20	21
Medium renewal	-	-	Y	-	Y	-	-	Y			-	-	Y	-	Y	-	Y	-	Y	-		Y	-	-	-
pH (new)	7.4	-	7.9	-	7.3	-	-	7.7			-	-	7.4	-	7.3	-	7.3	-	7.9	-		7.9	-	-	-
O2 (mg/l)      (new)	7.5	-	8.0	-	7.8	-	-	8.1			-	-	7.5	-	7.8	-	7.8	-	8.0	-		8.1	-	-	-
Temp (°C) (new)	18	-	21	-	20.7	-	-	18.7			-	-	19.1	-	20.9	-	20.7	-	18.2	-		18.9	-	-	-
Food provided	Y	-	Y	-	Y	-	-	Y			-	-	Y	-	Y	-	Y	-	Y	-		Y	-	-	-
Replicates	Number of living offspring per replicate																									Total no. of live offsprings per vessel
V1	-	-	-	-	-	-	-		-	-		-	11	-	-	-	29	--	-		25	19	19	-	-	84
V2	-	-	-	-	-	-	-		-	-		-	25	-	-	-	35	-	-		-	22	-	-	-	82
V3	-	-	-	-	-	-	-		-	-		-	-	18	-	8	-	37	-		-	15	-	-	-	78
V4	-	-	-	-	-	-	-		-	-		-	-	-	15	-	-	-	-		42	-	-	-	20	77
V5	-	-	-	-	-	-	-		-	-		-	16	-	-	41	-	1	-		-	14	-	-	-	72
V6	-	-	-	-	-	-	-		-	-		-	8	37	-	-	31	2	-		-	23	-	-	-	101
V7	-	-	-	-	-	-	-		-	-		-	-	8	-	-	31	-	-		-	30	1	-	-	70
V8	-	-	-	-	-	-	-		-	-		-	-	14	-	-	32	-	-		-	23	-	-	-	69
V9	-	-	-	-	-	-	-		-	-		-	21	-	-	-	30	24	-		-	15	-	-	-	90
V10	-	-	-	-	-	-	-		-	-		-	12	-	-	-	38	-	-		-	35	-	-	-	95
Cumulative Reproductivity																										818

 

No. of Days		0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
V1	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V2	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V3	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V4	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V5	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V6	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V7	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V8	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V9	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V10	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-

 

Nominal Concentration: 0.0625 mg/L

Day	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Medium renewal	-	-	Y	-	Y	-	-	Y	-	-	Y	-	Y	-	Y	-	Y	-	Y	-	-	-
pH (new)	8.0	-	8.0	-	7.8	-	-	7.6	-	-	8.1	-	7.3	-	7.5	-	7.4	-	7.5	-	-	-
O2 (mg/l)      (new)	7.7	-	8.2	-	8.1	-	-	8.0	-	-	7.6	-	7.6	-	7.9	-	7.5	-	7.4	-	-	-
Temp (°C) (new)	18	-	21	-	20.7	-	-	21.0	-	-	21.8	-	21.8	-	21.3	-	21.5	-	21.9	-	-	-
Food provided	Y	-	Y	-	Y	-	-	Y	-	-	Y	-	Y	-	Y	-	Y	-	Y	-	-	-
Replicates	Number of living offspring per replicate																						Total no. of live offspring’s per vessel
V1	-	-	-	-	-	-	-	-	-	-	-	-	18	-	-	-	-	35	-	17	-	1	71
V2	-	-	-	-	-	-	-	-	-	-	-	-	-	15	-	-	-	21	-	10	-	15	69
V3	-	-	-	-	-	-	-	-	-	-	11	-	-	-	31	-	-	31	1	1	-	19	93
V4	-	-	-	-	-	-	-	-	-	-	15	-	-	29	-	-	-	26	-	-	-	17	87
V5	-	-	-	-	-	-	-	-	-	-	15	-	-	16	-	-	-	9	-	11	-	21	72
V6	-	-	-	-	-	-	-	-	-	-	9	-	-	26	-	-	-	14	-	-	-	20	69
V7	-	-	-	-	-	-	-	-	-	-	10	-	-	-	27	-	-	16	-	9	-	22	84
V8	-	-	-	-	-	-	-	-	-	-	16	-	12	8	10	-	-	24	-	-	-	16	86
V9	-	-	-	-	-	-	-	-	-	-	13	-	-	-	25	-	-	27	-	-	-	-	65
V10	-	-	-	-	-	-	-	-	-	-	14	-	-	29	-	-	-	18	-	-	-	34	87
Cumulative Reproductivity																							775

 

 

 

No. of Days		0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
V1	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V2	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V3	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V4	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V5	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V6	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V7	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V8	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V9	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V10	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-

 

Nominal Concentration: 0.125 mg/L

Day	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Medium renewal	-	-	Y	-	Y	-	-	Y	-	-	Y	-	Y	-	Y	-	Y	-	Y	-	-	-
pH (new)	8.1	-	7.7	-	8.0	-	-	7.7	-	-	8.1	-	7.5	-	7.5	-	7.6	-	7.5	-	-	-
O2 (mg/l)      (new)	7.7	-	8.0	-	8.2	-	-	7.9	-	-	7.8	-	7.6	-	7.8	-	7.6	-	7.3	-	-	-
Temp (°C) (new)	18	-	21	-	20.7	-	-	21.0	-	-	21.8	-	21.8	-	21.3	-	21.5	-	21.9	-	-	-
Food provided	Y	-	Y	-	Y	-	-	Y	-	-	Y	-	Y	-	Y	-	Y	-	Y	-	-	-
Replicates	Number of living offspring per replicate																						Total no. of live offspring’s per vessel
V1	-	-	-	-	-	-	-	-	-	-	-	-	-	18	-	-	-	20	-	-	8	22	68
V2	-	-	-	-	-	-	-	-	-	-	15	-	-	29	-	-	-	14	-	-	-	18	100
V3	-	-	-	-	-	-	-	-	-	-	14	-	-	-	30	15	-	22	19	-	15	-	76
V4	-	-	-	-	-	-	-	-	-	-	10	-	-	11	9	-	-	12	5	-	-	8	70
V5	-	-	-	-	-	-	-	-	-	-	8	-	-	26	-	-	-	30	-	-	-	8	72
V6	-	-	-	-	-	-	-	-	-	-	15	-	29	-	23	-	-	17	-	-	-	-	91
V7	-	-	-	-	-	-	-	-	-	-	10	-	-	-	25	1	-	42	7	-	-	-	78
V8	-	-	-	-	-	-	-	-	-	-	-	-	-	16	-	-	-	34	-	-	-	17	67
V9	-	-	-	-	-	-	-	-	-	-	19	-	-	18	-	-	-	13	-	-	-	15	65
V10	-	-	-	-	-	-	-	-	-	-	11	-	-	22	-	-	-	21	-	-	-	16	70
Cumulative Reproductivity																							757

 

 

 

No. of Days		0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
V1	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V2	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V3	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V4	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V5	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V6	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V7	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V8	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V9	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V10	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-

 

Nominal Concentration: 0.25 mg/L

Day	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Medium renewal	-	-	Y	-	Y	-	-	Y	-	-	Y	-	Y	-	Y	-	Y	-	Y	-	-	-
pH (new)	8.2	-	7.9	-	7.9	-	-	7.7	-	-	8.2	-	7.5	-	7.6	-	7.5	-	7.4	-	-	-
O2 (mg/l)      (new)	7.7	-	7.9	-	8.3	-	-	8.0	-	-	7.9	-	7.6	-	8.0	-	7.7	-	7.3	-	-	-
Temp (°C) (new)	18	-	21	-	20.7	-	-	21.0	-	-	21.8	-	21.8	-	21.3	-	21.5	-	21.9	-	-	-
Food provided	Y	-	Y	-	Y	-	-	Y	-	-	Y	-	Y	-	Y	-	Y	-	Y	-	-	-
Replicates	Number of living offspring per replicate																						Total no. of live offspring’s per vessel
V1	-	-	-	-	-	-	-	-	-	-	19	-	-	25	-	-	-	25	-	-	-	17	86
V2	-	-	-	-	-	-	-	-	-	-	11	-	-	-	29	-	-	18	1	-	-	-	59
V3	-	-	-	-	-	-	-	-	-	-	11	-	-	23	-	1	-	25	-	-	-	34	94
V4	-	-	-	-	-	-	-	-	-	-	12	-	-	-	-	-	-	-	-	-	-	-	12
V5	-	-	-	-	-	-	-	-	-	-	19	-	-	35	-	-	-	15	-	-	-	19	88
V6	-	-	-	-	-	-	-	-	-	-	16	-	-	29	-	-	-	28	-	-	-	25	98
V7	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V8	-	-	-	-	-	-	-	-	-	-	11	-	-	28	-	-	-	30	-	-	-	24	93
V9	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V10	-	-	-	-	-	-	-	-	-	-	12	-	-	23	-	-	-	14	-	-	-	27	76
Cumulative Reproductivity																							606

 

 

 

No. of Days		0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
V1	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V2	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V3	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V4	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V5	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V6	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V7	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V8	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V9	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V10	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-

 

 

Nominal Concentration: 0.5 mg/L

Day	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20		21
Medium renewal	-	-	Y	-	Y	-	-	Y	-	-	Y	-	Y	-	Y	-	Y	-	Y	-	Y		-
pH (new)	8.1	-	8.0	-	8.0	-	-	7.9	-	-	8.2	-	7.6	-	7.8	-	7.6	-	7.6	-	7.8		-
O2 (mg/l)      (new)	7.6	-	8.0	-	8.1	-	-	8.0	-	-	7.7	-	7.5	-	7.7	-	7.7	-	7.4	-	7.8		-
Temp (°C) (new)	18	-	21	-	20.7	-	-	21.1	-	-	21.8	-	21.8	-	21.3	-	21.5	-	21.9	-	21.5		-
Food provided	Y	-	Y	-	Y	-	-	Y	-	-	Y	-	Y	-	Y	-	Y	-	Y	-	Y		-
Replicates	Number of living offspring per replicate																							Total no. of live offspring’s per vessel
V1	-	-	-	-	-	-	-	-	-	-	-	-	20	-	-	-	-	18	-	-	-	14		52
V2	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-		-
V3	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-		-
V4	-	-	-	-	-	-	-	-	-	-	-	-	18	-	-	-	-	27	-	27	-	-		72
V5	-	-	-	-	-	-	-	-	-	-	12	-	-	-	22	-	-	2	-	7	-	-		43
V6	-	-	-	-	-	-	-	-	-	-	-	-	-	10	10	-	-	22	22	-	-	19		41
V7	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-		-
V8	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-		-
V9	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-		-
V10	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	24	-	21	-	-		45
Cumulative Reproductivity																								236

 

 

 

No. of Days		0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
V1	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V2	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V3	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V4	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V5	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V6	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V7	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V8	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V9	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V10	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-

 

Nominal Concentration: 1 mg/L

Day	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Medium renewal	-	-	Y	-	Y	-	-	Y	-	-	Y	-	Y	-	Y	-	Y	-	Y	-	-	-
pH (new)	8.2	-	8.1	-	8.0	-	-	7.9	-	-	8.2	-	7.5	-	7.7	-	7.6	-	7.6	-	-	-
O2 (mg/l)      (new)	7.6	-	8.0	-	8.2	-	-	7.8	-	-	7.	-	7.6	-	7.5	-	7.8	-	7.3	-	-	-
Temp (°C) (new)	18	-	21	-	20.7	-	-	21.0	-	-	21.8	-	21.8	-	21.3	-	21.5	-	21.9	-	-	-
Food provided	Y	-	Y	-	Y	-	-	Y	-	-	Y	-	Y	-	Y	-	Y	-	Y	-	-	-
Replicates	Number of living offspring per replicate																						Total no. of live offspring’s per vessel
V1	-	-	-	-	-	-	-	-	-	-	-	-	3	1	-	-	-	21	-	-	-	12	37
V2	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V3	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V4	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V5	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V6	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V7	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V8	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V9	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V10	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Cumulative Reproductivity																							37

 

 

 

No. of Days		0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
V1	Dead P	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	M
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V2	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V3	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V4	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V5	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V6	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V7	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V8	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V9	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
V10	Dead P	-	-	-	-	-	M	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Dead O	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-

SIZE OF THE DAPHNIA AFTER 21 DAYS OF EXPOSURE

Concentrationmg/L	Size of the parent daphnia (cm)	Size of offspring's (cm)
Control	0.45	0.08
0.0625	0.40	0.08
0.125	0.39	0.08
0.25	0.35	0.05
0.5	0.30	0.05
1	0.32	0.05

Analytical monitoring: :

		Week 1		Week 2		Week 3
Sr. No.	Nominal conc. (mg/L)	Measured conc.		Measured conc.		Measured conc.
		New	Old	New	Old	New	Old
1	Blank
2	0.0625	0.070	0.070	0.066	0.063	0.068	0.062
3	0.125	0.124	0.110	0.114	0.101	0.112	0.110
4	0.25	0.247	0.203	0.223	0.235	0.215	0.218
5	0.5	0.489	0.404	0.462	0.437	0.436	0.453
6	1	0.993	0.966	0.967	0.993	0.903	0.980

Validity criteria fulfilled:

yes

Remarks:

No mortality was observed in control; Dissolved oxygen concentration >3 mg/L; Test chemical was maintained within 100±20% of test conc; Coefficent of variation in off spring production <25% between replicates; Avg number of offsprings per parent was 81.8;

Conclusions:

After 21 days of exposure tenure the NOEC was reported to be 0.25 mg/L, thus chemical can be caegorised into category 3 as per CLP classification criteria.

Executive summary:

Long term toxicity aquatic invertebrate:

Long term toxicity test to aquatic invertebrate was performed using OECD 211 study guidelines (Daphnia Magna Reproduction test). Test organisms used in the study was daphnia magna neonates of strain STRAUS. The study was conducted for the period of 21 days and number of living offsprings produced per living parent daphnia is calculated every day. The test chemical was dissolved in the M7 medium, stability study was conducted in M7 medium by dissolving 10 mg of test solution in 100 ml of medium which was kept under test conditions for 96 hours and sample at regular intervals of 24 hours was taken and checked for analytical stability. At 0.01 mg/l and 10 mg/L test chemical was with in the range of 100 ± 20% of initial test concentrations which was measured by using LCMS. Based on the stability results chemical semi static conditions was used in the study with a minimum renewal of three times a week. Gravid Parent Daphnids of age greater than 3 weeks was acclimitised for 2 days under test conditions. Offsprings obtained in the from second brood whose age < 24 hours was separated and exposed at different test concentrations for the main study. Based on the available data on short term toxicity test main study was conducted by taking concentrations of 0.0625, 0.125, 0.25, 0.5, and 1 mg/L, 0.5, and 1 mg/L along with one control group with M7 medium. 100 ml glass beakers were used with fill volume of 50 ml test solutions of different concentrations. Each test vessel was loaded with one offspring, for each test concentrations 10 replicates was used and each replicate has one daphnia, same was followed in control as well. Al beakers were checked for the pH and DO at every renewal day and recorded. The temperature, DO and pH in the control, 19 ± 0.5°C, day 0- 7.9 and day 21- 8.1, and Day 0 -7.5 and at Day 21 - 7.9 respectively. All parent daphnids were feed with freshwater algae (P. Subcapitata) during the time of renewal with quantity of 0.1-0.2 mg/carbon/daphnia. The photoperiod of 16 hours light and 8 hours dark conditions was maintained with light intensity 1221.75 lux. The number of living off springs produced per parent at each concentration was recorded, which is presented in the tables sections. The first offsprings observed on the control was on day 10. The average number of offspings produced per living parent was 81.8, 77.5, 75.75, 75.75, 52.6 and 37 in control, 0.0625, 0.125, 0.25, 0.5, and 1 mg/L respectively. Following the neonate production mortality of parent was also recorded, At concentration of 0.0625, 0.125, and control there was no mortality was observed in exposed populations, whereas, at concentrations of 0.25, 0.5, and 1 mg/L a percent of 20, 50 and 90 was observed. All exposure concentrations of pre and post renewal were analytical measured, using LCMS. Test chemical concentrations in old samples with concentrations 0.0625, 0.125, 0.25, 0.5, and 1 mg/L were in the range of 100.8 – 112, 80.8 -89.6, 81.2 – 94, 80.8 - 90.6, and 96.7 - 98.0%respectively. Test chemical concentrations in new samples with concentrations 0.0625, 0.125, 0.25, 0.5, and 1 mg/L were in the range of  105.6 – 112%, 89.6 - 99.2%, 86 - 98.8, 87.2 - 97.8 and 90.3 - 99.3 respectively. Which was found to be in the range of 100± 20% of initial test concentrations. All the validity crieria including the CV of offsprring production were with in the acceptibility range as per study guideline. Based on the outcomes, the NOEC was reported to be 0.25 mg/L considering the reproductive effects and LC50 is 0.46 mg/L considering the parent mortality. Thus chemical can be classified as category aquatic chronic category 2 as per CLP classification criteria.

Description of key information

Long term toxicity to aquatic invertebrate:

Long term toxicity test to aquatic invertebrate was performed using OECD 211 study guidelines (Daphnia Magna Reproduction test). Test organisms used in the study was daphnia magna neonates of strain STRAUS. The study was conducted for the period of 21 days and number of living offsprings produced per living parent daphnia is calculated every day. The test chemical was dissolved in the M7 medium, stability study was conducted in M7 medium by dissolving 10 mg of test solution in 100 ml of medium which was kept under test conditions for 96 hours and sample at regular intervals of 24 hours was taken and checked for analytical stability. At 0.01 mg/l and 10 mg/L test chemical was with in the range of 100 ± 20% of initial test concentrations which was measured by using LCMS. Based on the stability results chemical semi static conditions was used in the study with a minimum renewal of three times a week. Gravid Parent Daphnids of age greater than 3 weeks was acclimitised for 2 days under test conditions. Offsprings obtained in the from second brood whose age < 24 hours was separated and exposed at different test concentrations for the main study. Based on the available data on short term toxicity test main study was conducted by taking concentrations of 0.0625, 0.125, 0.25, 0.5, and 1 mg/L, 0.5, and 1 mg/L along with one control group with M7 medium. 100 ml glass beakers were used with fill volume of 50 ml test solutions of different concentrations. Each test vessel was loaded with one offspring, for each test concentrations 10 replicates was used and each replicate has one daphnia, same was followed in control as well. Al beakers were checked for the pH and DO at every renewal day and recorded. The temperature, DO and pH in the control, 19 ± 0.5°C, day 0- 7.9 and day 21- 8.1, and Day 0 -7.5 and at Day 21 - 7.9 respectively. All parent daphnids were feed with freshwater algae (P. Subcapitata) during the time of renewal with quantity of 0.1-0.2 mg/carbon/daphnia. The photoperiod of 16 hours light and 8 hours dark conditions was maintained with light intensity 1221.75 lux. The number of living off springs produced per parent at each concentration was recorded, which is presented in the tables sections. The first offsprings observed on the control was on day 10. The average number of offspings produced per living parent was 81.8, 77.5, 75.75, 75.75, 52.6 and 37 in control, 0.0625, 0.125, 0.25, 0.5, and 1 mg/L respectively. Following the neonate production mortality of parent was also recorded, At concentration of 0.0625, 0.125, and control there was no mortality was observed in exposed populations, whereas, at concentrations of 0.25, 0.5, and 1 mg/L a percent of 20, 50 and 90 was observed. All exposure concentrations of pre and post renewal were analytical measured, using LCMS. Test chemical concentrations in old samples with concentrations 0.0625, 0.125, 0.25, 0.5, and 1 mg/L were in the range of 100.8 – 112, 80.8 -89.6, 81.2 – 94, 80.8 - 90.6, and 96.7 - 98.0%respectively. Test chemical concentrations in new samples with concentrations 0.0625, 0.125, 0.25, 0.5, and 1 mg/L were in the range of  105.6 – 112%, 89.6 - 99.2%, 86 - 98.8, 87.2 - 97.8 and 90.3 - 99.3 respectively. Which was found to be in the range of 100± 20% of initial test concentrations. All the validity crieria including the CV of offsprring production were with in the acceptibility range as per study guideline. Based on the outcomes, the NOEC was reported to be 0.25 mg/L considering the reproductive effects and LC50 is 0.46 mg/L considering the parent mortality. Thus chemical can be classified as category aquatic chronic category 2 as per CLP classification criteria.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.25 mg/L

Additional information

Long term toxicity to aquatic invertebrate:

Data available for the structurally and functionally read across chemicals has been reviewed to determine the long term toxicity of test chemical to aquatic invertebrates. The results of studies are as summarized below:

 

Long term toxicity test to aquatic invertebrate was performed using OECD 211 study guidelines (Daphnia Magna Reproduction test). Test organisms used in the study was daphnia magna neonates of strain STRAUS. The study was conducted for the period of 21 days and number of living offsprings produced per living parent daphnia is calculated every day. The test chemical was dissolved in the M7 medium, stability study was conducted in M7 medium by dissolving 10 mg of test solution in 100 ml of medium which was kept under test conditions for 96 hours and sample at regular intervals of 24 hours was taken and checked for analytical stability. At 0.01 mg/l and 10 mg/L test chemical was with in the range of 100 ± 20% of initial test concentrations which was measured by using LCMS. Based on the stability results chemical semi static conditions was used in the study with a minimum renewal of three times a week. Gravid Parent Daphnids of age greater than 3 weeks was acclimitised for 2 days under test conditions. Offsprings obtained in the from second brood whose age < 24 hours was separated and exposed at different test concentrations for the main study. Based on the available data on short term toxicity test main study was conducted by taking concentrations of 0.0625, 0.125, 0.25, 0.5, and 1 mg/L, 0.5, and 1 mg/L along with one control group with M7 medium. 100 ml glass beakers were used with fill volume of 50 ml test solutions of different concentrations. Each test vessel was loaded with one offspring, for each test concentrations 10 replicates was used and each replicate has one daphnia, same was followed in control as well. Al beakers were checked for the pH and DO at every renewal day and recorded. The temperature, DO and pH in the control, 19 ± 0.5°C, day 0- 7.9 and day 21- 8.1, and Day 0 -7.5 and at Day 21 - 7.9 respectively. All parent daphnids were feed with freshwater algae (P. Subcapitata) during the time of renewal with quantity of 0.1-0.2 mg/carbon/daphnia. The photoperiod of 16 hours light and 8 hours dark conditions was maintained with light intensity 1221.75 lux. The number of living off springs produced per parent at each concentration was recorded, which is presented in the tables sections. The first offsprings observed on the control was on day 10. The average number of offspings produced per living parent was 81.8, 77.5, 75.75, 75.75, 52.6 and 37 in control, 0.0625, 0.125, 0.25, 0.5, and 1 mg/L respectively. Following the neonate production mortality of parent was also recorded, At concentration of 0.0625, 0.125, and control there was no mortality was observed in exposed populations, whereas, at concentrations of 0.25, 0.5, and 1 mg/L a percent of 20, 50 and 90 was observed. All exposure concentrations of pre and post renewal were analytical measured, using LCMS. Test chemical concentrations in old samples with concentrations 0.0625, 0.125, 0.25, 0.5, and 1 mg/L were in the range of 100.8 – 112, 80.8 -89.6, 81.2 – 94, 80.8 - 90.6, and 96.7 - 98.0%respectively. Test chemical concentrations in new samples with concentrations 0.0625, 0.125, 0.25, 0.5, and 1 mg/L were in the range of  105.6 – 112%, 89.6 - 99.2%, 86 - 98.8, 87.2 - 97.8 and 90.3 - 99.3 respectively. Which was found to be in the range of 100± 20% of initial test concentrations. All the validity crieria including the CV of offsprring production were with in the acceptibility range as per study guideline. Based on the outcomes, the NOEC was reported to be 0.25 mg/L considering the reproductive effects and LC50 is 0.46 mg/L considering the parent mortality. Thus chemical can be classified as category aquatic chronic category 2 as per CLP classification criteria.

In first study an experiment was performed to determine the Long term toxicity effect on reproduction of Daphnia magna for 20 days as per OECD Guideline 211 (Daphnia magna Reproduction Test). In this experiment Daphnia magna less then 24h old were used at the initiation of the test. They were exposed to various concentrations of test chemicals .The test medium used was water which is obtained from 33% evian water and 67% volvic water which are commonly marked as mineral water in Japan and it was changed every 1 or 2 days, neonatal were counted every day. The total number of fleas born over 21 days in the test fluid of each concentration and the total number born in the control group were statistically processed to determine NOEC and LOEC values.

After 21 days, of exposure of test chemical to Daphnia magna the NOEC was observed to be 0.125 mg/l and LOEC value was determined to be 0.25 mg/L .On the basis of above effect concentration it can be considered that test material has chronic effects on aquatic invertebrate and can be classified as aquatic chronic 2 as per CLP criteria.

 

Another study was conducted to access the effect of test chemical on reproduction of aquatic invertebrates Daphnia magna after the exposure of test chemical for 21 days. This study was conducted in accordance with the OECD Guideline 211 (Daphnia magna Reproduction Test). After 21 days of exposure of test chemical to daphnia magna the NOEC value was determined to be 0.98 mg/L and EC50 value was determined to be in range of 0.98- 2.7 mg/L, On the basis of these values test chemical is considered to be toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

 

By considering results of both the studies mentioned above and applying weight of evidence approach the NOEC value of test chemical was determined to be in range from 0.125 mg/L to 0.98 mg/l after exposure of test chemical for 21 days. The range NOEC value indicates that the substance is likely to be toxic to aquatic algae and can be classified as aquatic chronic 2 per the CLP classification criteria.