[No public or meaningful name is available]

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2016 - July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL:
- Chemical name: Phosphonium, tetrakis (hydroxymethyl)-,sulphate(2:1); polymer with urea
- CAS number: 63502-25-0
- Batch no.: PLFS17D1
- Expiration date of the lot/batch: 31. August 2016
- Purity: Active content (Iodine titration): 67.8% w/w / THPS (CAS number 55566-30-8): 25.7% w/w
- Solubility in water: Miscible
- pH: 5.0 at 20 °C
- Specific gravity (s.g.): 1.377 g/ml
- Appearance: Clear, yellow liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
- Stability under correct storage conditions (expiry date): 31. August 2016
- Storage requirements: Keep container tightly closed in a cool, dry place.

Analytical monitoring:

yes

Details on sampling:

- Sampling method:
Samples of stock and/or test solutions were taken at the start and at the end of exposure (i.e. T0 hours and T48 hours) to determine the actual test item concentrations. Control and test solutions will be sampled in duplicate. The duplicate samples will be kept separately as a reserve.

- Sample storage conditions before analysis:
After sampling and before shipment, the samples will be stored in glass bottles in the dark at a temperature of ≤ -18°C if not advised otherwise.
Samples were transferred to the test site for chemical analysis under the required storage conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
A stock solution (S1) of the test item was prepared by dissolving 1.4767 g of the test item in 10000 mL of test medium under stirring, resulting in a nominal concentration of 147 mg test item/L (100 mg a.i./L). This stock solution was stirred for 10 minutes at ambient temperature. The stock solution was used immediately to prepare the desired test item concentrations by serial dilution. The stock solution was used, undiluted, as the highest test concentration. The diluted test solutions were stirred for another 2 min and were stored at test temperature in the dark for the duration of the test.
During dosing into the flow-through system, the test solutions were stored in reservoir glass tanks covered in aluminium foil at test temperature, and were placed near the testing area

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: STRAUS (clone M10),
- Source: internal stock culture (originally supplied by KU Leuven, Belgium)
- Age of parental stock (mean and range, SD): parental daphnids aged less than 35 days
- Age at study initiation: young (6-24 h)
- Feeding during test: none

ACCLIMATION / HOLDING CONDITION
- Acclimation conditions: no as the specimens are cultured in same conditions as the tests,i.e. 16/8 hours light/dark cycle; light intensity is 50–1000 lx; the temperature is 20±2 °C
- Type and amount of food: The daphnids are fed with e.g. Algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS:
The holding vessels designated for production of the neonates for the test will be cleared of all mixed-aged young daphnids on the day before test start. These vessels will contain parental daphnids aged less than 35 days. On the day of test start (day 0) all newly produced neonates of these vessels will be < 24 hours old. These neonates will be collected for the test.
The young daphnids were transferred into the test solutions by using an upside-down Pasteur-pipette. This was done in a randomised manner, e.g. by sequentially adding one daphnid per test vessel until all test vessels contain five mobile daphnids.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

14.2 °dH

Test temperature:

between 20.2 and 20.9°C

pH:

between 6.9 and 7.9

Dissolved oxygen:

between 8.6 and 8.8 mg/L

Salinity:

254 mg/L CaCO3

Conductivity:

672 µS/cm

Nominal and measured concentrations:

Based on the results of a preliminary non-GLP range finding test, the following nominal concentrations in a geometrical series (spacing factor: 2.2) were tested in the definitive test:
- 100, 45.5, 20.7, 9.39 and 4.27 mg active ingredient/L.

These concentrations correspond to the following concentrations of test item, using the content of active ingredient of 67.8% (w/w) as reported in the Certificate of Analysis (see also section 10.1):
- 147, 67.0, 30.5, 13.9 and 6.30 mg test item/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (e.g. 300 mL) covered e.g. with watch glasses will be used as test vessels.
- Type : open
- Material, size, headspace, fill volume: Each test vessel will contain 150 to 200 mL of test solution.
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): peristaltic pump: Type Ismatec ISM 40 AB, Ismatec, Wertheim Mohnfeld, Germany, including tubing type: TYGON SI.
- Renewal rate of test solution (frequency/flow rate): flow-through regimen (ca. 5 vessel volumes per day)
- No. of organisms per vessel: 5 dpahnids
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per control (replicates): 4 replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt Medium M4
The required volume of medium was prepared within one month before use. During storage at ambient temperature, the medium will be aerated. The physical-chemical characteristics (pH, hardness, oxygen, conductivity, alkalinity and temperature) of the Medium M4 were determined before use.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 hours light/dark cycle.
- Light intensity: 50 to 1000 lx.

EFFECT PARAMETERS MEASURED
At start of the test, after temperature adaptation of the test solutions, and at the end of the test the following parameters were measured and recorded in one replicate test vessel of the control and the highest test concentration
- temperature, pH and dissolved oxygen content in % of air saturation value (ASV) and in mg/L.


RANGE-FINDING STUDY
- Range finding study: yes
- Test concentrations (range-finding test): 0.16, 0.80, 4.00, 20, 100 mg active ingredient/L, These concentrations correspond to the following concentrations of test item, using the content of active ingredient of 67.8% (w/w): 0.236, 1.18, 5.90, 29.5, 147.95 mg test item/L.

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

7.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other:

Remarks:

THPS

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

18.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

27.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

1.81 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other:

Remarks:

THPS

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

4.78 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

7.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST:
No immobilization was observed at 0.16, 0.80, 4.00, 20 mg active ingredient/L (except one daphnid in the group 4.00) . However, 100% immobilization was observed at 100 mg active ingredient/L

DEFINITIVE TEST:
- Physico-chemical parameters: see table 2 in "any other information on results including tables"


- Immobilization Data and sublethal observations: see table 3 in "any other information on results including tables"
There were no death in the control ann 100% of death in the highest test concentration. A summary of the immobility data for Daphnia magna are shown in table 2 in "any other information on results including tables)
Sublethal observations on the mobile daphnids were recorded after 24 and 48 h of exposure at all concentration levels. An increased number of floaters, and reduced swimming activity were observed at 67.0 and 147 mg test item/L.

- Analytical finding: see table 4 in "any other information on results including tables"
Samples from the test solutions were taken to determine actual levels of the active ingredient in comparison to nominal concentrations. The test item being a multi-constituent, measured concentrations of THPS, its most unstable constituent (nominal 25.7% of test item as per Certificate of Analysis), were used as quantifiers for the content of the test item in the test medium.
The recoveries based on geometric mean measured THPS concentrations (% of nominal THPS concentrations) determined during the test were between 19.6 and 94.4% of nominal.

A clear concentration-response relationship was observed. Some measured concentrations being below 80% of nominal concentrations, the ECx must be based on the geometric mean of the measured concentrations.
Under the conditions of the present study, the 48h-EC50 was 7.12 mg/L expressed as THPS/L corresponding to 18.8 mgL expressed as active ingredient and 27.7 mg/L expressed as test item (see table 5 "any other information on results including tables)

Results with reference substance (positive control):

- Results with reference substance valid: yes
The most recent reference test with potassium dichromate performed in a separate study resulted in an EC50(24h) of 1.68 mg/L. According to the OECD guideline No. 202 (OECD, April 2004) EC50(24 h)-values for potassium dichromate obtained from different laboratories should range between 0.6 mg/L and 2.1 mg/L.

Reported statistics and error estimates:

. Weibull analysis using linear maximum likelihood regression was used to estimate the ECx values.
The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.

Table 1: Results of the non-GLP rangefinding test under flow-through conditions.

	Perform LF	THPS urea monomer	Mobile daphnids
Treatment	[mg/L]	[mg/L]	0 h	24 h	48 h
Control	0	0	5	5	5
C1	0.236	0.160	5	5	5
C2	1.18	0.800	5	5	5
C3	5.90	4.00	5	5	4
C4	29.5	20.0	5	5	5
C5	147.49	100	5	0	0

Table 2: Summary of physico-chemical parameters in the test solutions

parameter	unit	minimum	maximum	n
pH	-	6.9	7.9	12
temperature	[°C]	20.2	20.9	4
oxygen	[% of air saturation]	95	99	4
oxygen	[mg/L]	8.6	8.8	4
light intensity	[lx]	234	756	4

Table 3: Cumulative number of immobilised Daphnia magna after 24 and 48 h.

nominal concentration	total number of introduced daphnids	total number of immobilised daphnids	immobilised daphnids (% of introduced)	total number of immobilised daphnids	immobilised daphnids (% of introduced)
[mg test item/L]		[24 h]	[24 h]	[48 h]	[48 h]
Control	20	0	0.0	0	0.0
6.30	20	1	5.0	2	10.0
13.9	20	1	5.0	1	5.0
30.5	20	1	5.0	2	10.0
67.0	20	1	5.0	19	95.0
147	20	5	25.0	20	100.0

Table 4 : Summary of measured concentrations during the test: Measured THPS concentrations, geometric mean and % of nominal

Nominal concentration [mg test item/L]	Nominal concentration [mg active ingredient/L]	Nominal concentration THPS [mg THPS/L]	Test period [h]	Measured concentration [mg THPS/L]	Recovery in % of nominal THPS	Measured concentration [mg THPO/L]	Geometric mean measured concentrations
							[mg THPS/L]	in % of nominal THPS
0 (control)	0	0	0	n.d.	n.a.	n.d.
6.30	4.27	1.62	0	0.19	11.7	0.80
13.9	9.4	3.57	0	0.65	18.2	1.66
30.5	20.7	7.84	0	4.93	62.9	1.86
67	45.4	17.22	0	14.4	83.6	5.09
147	100	37.8	0	37.2	98.5	2.67
0 (control)	0	0	48	n.d.	n.a.	n.d.	n.a.	n.a.
6.30	4.27	1.62	48	0.53	32.7	0.62	0.317	19.6
13.9	9.42	3.57	48	1.72	48.1	1.70	1.06	29.6
30.5	20.7	7.84	48	5.66	72.2	1.53	5.28	67.4
67	45.4	17.22	48	14.9	86.5	4.99	14.65	85.1
147	100	37.8	48	34.2	90.5	3.45	35.7	94.4

Table 5: EC_xvalues of the parameter "immobility of daphnids" (48 h) based on mean measured concentrations in mg test item/L, mg a.i./L (67.8% of test item) and mg THPS/L (25.7% of test item).

immobility of daphnids (48 h)	EC10	EC20	EC50
	[mg test item/L]
	7.04	12.1	27.7
lower 95%-cl	n.d.	n.d.	n.d.
upper 95%-cl	n.d.	n.d.	n.d.
	[mg a.i./L]
	4.78	8.2	18.8
lower 95%-cl	n.d.	n.d.	n.d.
upper 95%-cl	n.d.	n.d.	n.d.
	[mg THPS/L]
	1.81	3.12	7.12
lower 95%-cl	n.d.	n.d.	n.d.
upper 95%-cl	n.d.	n.d.	n.d.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, the 48h-EC50 was 7.12 mg/L expressed as THPS/L corresponding to 18.8 mgL expressed as active ingredient and 27.7 mg/L expressed as test item.

Executive summary:

The acute immobilization (EC50 and NOEC) of the Reaction mass towards Daphnia magna was determined in a 48‑hour static test according to the OECD Guidelines for Testing of Chemicals (April 2004) No 202,"Daphnia sp., Acute Immobilisation Test". The study was compliant with the GLP.

Based on a preliminary range-finding test, twenty daphnids were exposed to the test item over a range of nominal concentrations of 100, 45.5, 20.7, 9.39 and 4.27 mg active ingredient/L corresponding to 147, 67.0, 30.5, 13.9 and 6.30 mg test item/L and control for 48 hours under flow-through conditions. The 20 daphnids per test solution, divided into four replicates of 5 daphnids each, were observed for immobility after 24 and 48 hours of exposure

According to the state of art of the analytical method, only two component of the reaction mass could be monitored; the THPS and the THPO (the last one corresponding to a degradation product). Therefore, measured concentrations of THPS were used as quantifiers for the content of the test item in the test medium.

The recoveries based on geometric mean measured THPS concentrations (% of nominal THPS concentrations) determined during the test were between 19.6 and 94.4% of nominal (at the lowest concentration and the highest concentrations respectively).

 

A clear concentration-response relationship was observed.

The EC50 values of the parameter "immobility of daphnids" at 48 h based on the on statistical evaluation of biological results and nominal concentrations was 40.6 mg/L expressed as test item corresponding to 27.5 mg/L expressed as active ingredient (67.8% of test item).

The EC50 values of the parameter "immobility of daphnids" at 48 h based on the on statistical evaluation of biological results and the geometric mean of measured concentrations of THPS was 7.12 mg/L expressed as THPS/L corresponding to 18.8 mgL expressed as active ingredient and 27.7 mg/L expressed as test item.

The two validity criteria of the OECD guideline 202 were fulfilled, therefore this study is considered as reliable without restrictions

 

Description of key information

One reliable study is available for the Reaction mass THPS-urea monomer for this endpoint.

This Acute Toxicity Study was conducted in order to assess the effect of the Reaction mass THPS-urea monomer on the

mobility of Daphnia magna during an exposure period of 48 hours.

It was performed according to the standard OECD 202 guideline and was GLP compliant.

Based on a preliminary range-finding test, twenty daphnids were exposed to the test item over a range of nominal concentrations of 100, 45.5, 20.7, 9.39 and 4.27 mg active ingredient/L corresponding to 147, 67.0, 30.5, 13.9 and 6.30 mg test item/L and control for 48 hours under flow-through conditions. The 20 daphnids per test solution, divided into four replicates of 5 daphnids each, were observed for immobility after 24 and 48 hours of exposure.

According to the state of art of the analytical method, only two component of the reaction mass could be monitored; the THPS and the THPO (the last one corresponding to a degradation product). Therefore, measured concentrations of THPS were used as quantifiers for the content of the test item in the test medium.

The recoveries based on geometric mean measured THPS concentrations (% of nominal THPS concentrations) determined during the test were between 19.6 and 94.4% of nominal (at the lowest concentration and the highest concentrations respectively).

 

A clear concentration-response relationship was observed.

The EC50 values of the parameter "immobility of daphnids" at 48h based on the on statistical evaluation of biological results and nominal concentrations was 40.6 mg/L expressed as test item corresponding to 27.5 mg/L expressed as active ingredient (67.8% of test item).

The EC50 values of the parameter "immobility of daphnids" at 48h based on the on statistical evaluation of biological results and the geometric mean of measured concentrations of THPS was 7.12 mg/L expressed as THPS/L corresponding to 18.8 mg/L expressed as active ingredient and 27.7 mg/L expressed as test item.

 

Therefore, as only the THPS concentrations could be measured and as a conservative result, the EC50 of the registered reaction mass is based on the geometric mean of measured concentrations of THPS i.e. 48h-EC50 = 18.8 mg/L expressed as active ingredient.

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

18.8 mg/L