Reaction products of disubstituted alkane, nickel, (Z/E)-substituted alkene, reaction mass of tetraalkyl-3,3’-di(propan-2-yl)-biphenyl-diyl tetrakis(dialkylphenyl) bis(phosphite) and tris(dialkylphenyl) phosphite and zinc halide

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:

2018-10-03 to 2018-11-26

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)

Version / remarks:

Adopted April 13, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 23, Guidance document on aquatic toxicity testing of difficult substances and mixtures

Version / remarks:

2000

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Frequency:
At the start (t=0 h) and the end of the test (t=48 h).
Concentrations:
Samples for analysis were taken from all concentrations and the control, immediately before transfer to the individual replicates. Care was taken not to include any floating layer, test item film or undissolved material in separate vessels.
At the end of the test, samples were taken from the approximate center of the pooled solutions of the vessels at each concentration.
Number of samples:
Sampling consisted of single samples per treatment.
Volume:
2 mL per sample;
Storage:
Samples were stored in a freezer (=-15°C) until analysis.

Vehicle:

no

Details on test solutions:

Water Accommodated Fractions were prepared:
Individual loading rates were vigorously agitated on a magnetic stirrer for 6 days. On day 3 of stirring, pH was measured in case adjustment would be necessary (no adjustment was necessary: pH of 8.0 ± 0.3). After 6 days of stirring, the obtained mixtures were allowed to settle for 1 day in order to allow separation of phases. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning over glasswool (put into a funnel). Glasswool filtering was necessary to avoid a positive Tyndall effect. Glasswool was pre-saturated with the test item beforehand. No Tyndall effect was observed in any prepared solution, and solutions were clear and colourless at the end of the preparation procedure.
Thereafter, the aqueous WAFs were used as test concentrations.
Based on preliminary results, the period of 6 days of stirring was used to ensure that
a) Concentration of the relevant transition metal stabilizes at the pH 8.0 ± 0.3 used for ecotoxicity testing to prevent any precipitation during the test;
b) Saturation of the aquatic phase with organic hydrolysis products stemming from aromatic phosphites is obtained.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress (such as mortality >20%, presence of males, ephippia or discolored animals) and there was no delay
in the production of the first brood.
Characteristics Daphnia: less than 24 hours old, from parental daphnids of more than two weeks old.

BREEDING
Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, are placed into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Culture medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The hardness of test medium expressed as CaCO3: 180 mg/L

Test temperature:

20 °C (± 1 °C)

pH:

pH of 8.0 ± 0.3.

Dissolved oxygen:

= 3 mg/L

Salinity:

not applicable

Nominal and measured concentrations:

A combined range-finding and limit test was performed, with the following individually prepared water accommodated fractions (WAF, based on nominal loading rate):
1.0 mg/L; 10 mg/L; 100 mg/L
Please see IUCLID section "Any other information on results incl. tables" below for information on analytical results.

Details on test conditions:

Please see IUCLID section "Any other information on materials and methods incl. tables" below for details.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL0

Remarks:

Based on Water Accommodated Fraction (WAF) approach

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL10

Remarks:

Based on Water Accommodated Fraction (WAF) approach

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL50

Remarks:

Based on Water Accommodated Fraction (WAF) approach

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

See IUCLID section "Any other information on results incl. tables" for details.

Results with reference substance (positive control):

The 24 h EC50 for potassium dichromate was in the recommended range of 0.6 to 2.1 mg/L according to OECD 202.

Reported statistics and error estimates:

Not applicable - no effects observed.

These data are based on results from draft report and will be amended as soon as the final report will have been issued.

Details on Biological Data:

No immobility was observed in the control and at any test concentration throughout the test (see table below).

Time (h)	Replicate	Step 2 Catalyst (Stripped) Loading rate (mg/L)
		Control	1.0	10	100
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0	0 (1)	0 (1)	0
	B	0	0	0	0
	C	0			0
	D	0			0
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0
48	A	0	0	0	0
	B	0	0	0	0
	C	0			0
	D	0			0
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Details on Analytical Data

1) Analysis of the relevant transition metal by ICP-MS

Time of sampling hours	Date of sampling	Date of analysis	Loading rate (WAF) [mg/L]	Concentration test item analysed [mg/L]	Relative to initial [%]	Transition metal concentration calculated from elemental fraction (0.8%) determined in study number 519586 [mg/L]
0	15 Oct 2018	24 Oct 2018	0	-0.0092^1	--	0.000
			100	23.6	--	0.189
48	17 Oct 2018	24 Oct 2018	0	-0.024^1	n.a.	0.000
			100	23.5	99	0.188

(1) Estimated value, calculated by extrapolation of the calibration curve. The value is lower than the intercept so the analyzed concentration is a negative value

2) Analysis of the organics fraction by LC-UV at 210 nm

Time of sampling [hours]	Date of sampling	Date ofanalysis	Loading rate WAF [mg/L]	Concentration analysed [mg/L]	Relative to initial [%]
0	15 Oct 2018	31 Oct 2018	0	n.d.
			100	126
48	17 Oct 2018	31 Oct 2018	0	0.023
			100	157	125

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity to aquatic invertebrates was tested according to OECD 202 compliant with GLP. Due to the nature of Step 2 catalyst (UVCB), according to OECD 23 water accommodated fractions (WAF) were prepared.
Result: EL0 (Daphnia magna; 48 h; static) = 100 mg/L;
EL10 (Daphnia magna; 48 h; static) > 100 mg/L;
EL50 (Daphnia magna; 48 h; static) > 100 mg/L

Executive summary:

The acute toxicity to aquatic invertebrates was tested according to OECD 202 compliant with GLP. The test organism was Daphnia magna (Straus). Due to the nature of Step 2 catalyst (UVCB), according to OECD 23 water accommodated fractions (WAF) were prepared and a combined range finding and limit test conducted. WAFs were prepared at 1, 10 and 100 mg/L nominal loading: individual loading rates were vigorously agitated on a magnetic stirrer for 6 days. Based on preliminary results, the period of 6 days of stirring was used to ensure that

a) Concentration of the relevant transition metal stabilizes at the pH 8.0 ± 0.3 used for ecotoxicity testing to prevent any precipitation during the test;

b) Saturation of the aquatic phase with organic hydrolysis products stemming from aromatic phosphites is obtained.

The presence of any non-dissolved test item fraction could be excluded before testing by measuring the Tyndall effect.

Analytical monitoring was performed for the relevant transition metal (ICP-MS) as well as the organic fraction (UPLC-UV at 210 nm). At the limit concentration of WAF 100 mg/L transition metal concentration was stable over the 48 hours of the test. A Step 2 catalyst concentration equivalent of 23.6 mg/L was found in freshly prepared WAF (after 6 days of stirring and one day settling period) and 99% of this initial concentration was recovered after 48 hours (23.5 mg/L). This is equivalent to transition metal concentrations at 0 h and 48 hours of 189 µg/L and 188 µg/L, respectively.

With regard to the organic fraction monitored via UPLC-UV at 210 nm, an initial Step 2 catalyst concentration equivalent of 126 mg/L was measured at the limit concentration of WAF 100 mg/L. This increased during 48 hours of incubation by 25%. The reason for this increase is not clear. It could be due to further abiotic transformations (hydrolysis / oxidation) during the test. The results confirm in any case that WAF preparation succeeded in a relevant organic fraction present at the beginning and during the test.  

For all concentrations tested (including the limit concentration of WAF 100 mg/L) neither in control groups nor in treatment groups any immobilizations or other effects could be determined.

Result:

Result: EL0 (Daphnia magna; 48 h; static) = 100 mg/L;

EL10 (Daphnia magna; 48 h; static) > 100 mg/L;

EL50 (Daphnia magna; 48 h; static) > 100 mg/L

Description of key information

The acute toxicity to aquatic invertebrates was tested according to OECD 202 compliant with GLP. Due to the nature of Step 2 catalyst (UVCB), according to OECD 23 water accommodated fractions (WAF) were prepared.

Result:

EL0 (Daphnia magna; 48 h; static) = 100 mg/L;

EL10 (Daphnia magna; 48 h; static) > 100 mg/L

EL50 (Daphnia magna; 48 h; static) > 100 mg/L

Additional information

The acute toxicity to aquatic invertebrates was tested according to OECD 202 compliant with GLP. The test organism was Daphnia magna (Straus). Due to the nature of Step 2 catalyst (UVCB),

according to OECD 23 water accommodated fractions (WAF) were prepared and a combined range finding and limit test conducted. WAFs were prepared at 1, 10 and 100 mg/L nominal loading:

individual loading rates were vigorously agitated on a magnetic stirrer for 6 days. Based on preliminary results, the period of 6 days of stirring was used to ensure that

a) Concentration of the relevant transition metal stabilizes at the pH 8.0 ± 0.3 used for ecotoxicity testing to prevent any precipitation during the test;

b) Saturation of the aquatic phase with organic hydrolysis products stemming from aromatic phosphites is obtained.

The presence of any non-dissolved test item fraction could be excluded before testing by measuring the Tyndall effect.

Analytical monitoring was performed for the relevant transition metal (ICP-MS) as well as the organic fraction (UPLC-UV at 210 nm). At the limit concentration of WAF 100 mg/L transition

metal concentration was stable over the 48 hours of the test. A Step 2 catalyst concentration equivalent of 23.6 mg/L was found in freshly prepared WAF (after 6 days of stirring and one day

settling period) and 99% of this initial concentration was recovered after 48 hours (23.5 mg/L). This is equivalent to transition metal concentrations at 0 h and 48 hours of 189 µg/L and

188 µg/L, respectively.

With regard to the organic fraction monitored via UPLC-UV at 210 nm, an initial Step 2 catalyst concentration equivalent of 126 mg/L was measured at the limit concentration of WAF 100 mg/L.

This increased during 48 hours of incubation by 25%. The reason for this increase is not clear. It could be due to further abiotic transformations (hydrolysis / oxidation) during the test.

The results confirm in any case that WAF preparation succeeded in a relevant organic fraction present at the beginning and during the test.  

For all concentrations tested (including the limit concentration of WAF 100 mg/L) neither in control groups nor in treatment groups any immobilizations or other effects could be determined.

Result:

EL0 (Daphnia magna; 48 h; static) = 100 mg/L;

EL10 (Daphnia magna; 48 h; static) > 100 mg/L;

EL50 (Daphnia magna; 48 h; static) > 100 mg/L