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Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

08.03.07 - 16.03.07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ISO 14669; 1999

Version / remarks:

Water Quality - Determination of acute lethal toxicity to marine copepods
(Copepoda, Crustacea)

Principles of method if other than guideline:

This test was judged to be valid according to the following criteria as stated In test guideline
ISO 14669, 1999:
• Dissolved O2 concentration in the test medium was >4 mg r l at the end ofthe test
• Percentage mortality in the controls was ~l 0%
• Mortality after 48 h in the presence of a standard reference toxicant (1.0 mg r l 3,5,dichlorophenol)
was in the range 20 - 80%

GLP compliance:

yes

Specific details on test material used for the study:

Test item name: Dodicor V 5654
Batch/Ref ID No.: 07-YS-019
Manufacturer/Supplier: Clariant GmbH
Date test item received: 12.02.07
Study Ref No.: 2221/acartia

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

other: Acartia Tonsa (Dana)

Details on test organisms:

Acartia tonsa (Dana), originally from laboratory cultures maintained at the Laboratory of
Marine Biology, 3000 Helsing0r, DenI11ark, are cultured in facilities at Clariant Oil Services.
Eggs are hatched and stock cultures of Acartia tonsa are maintained in aerated seawater in 10litre
round, glass flasks at the test temperature of 20±2°C. Illumination is provided by cool,
white fluorescent lights on a 12-h light, 12-h dark cycle. Cultures are fed on cultured algal cells
(Rhodomonas baltica).

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 +- 2 °C

pH:

Start: 4.84 - 8.19
End 5.50 - 8.14

Salinity:

Natural Seawater

Nominal and measured concentrations:

27.6 - 81.6 - 240 - 727 - 2204 mg/L and control

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

354 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Validity criteria fulfilled:

yes

Conclusions:

An acute lethality test was performed with Acartia tansa (Dana) using Dodicor V 5654 at nominal
concentrations of 27.6- 2204 mg rl. The following results were obtained:
48-h LC50 : 354 mg/L
24-h LC50: 523 mg/L
48-h LC100: 696 mg(L
48-hNOEC: < 27.6 mg/L

Executive summary:

An acute lethality test was performed withAcartia tansa(Dana) using Dodicor V 5654 at nominal

concentrations of 27.6- 2204 mg rl. The following results were obtained:

48-hLC50 : 354 mg/L

24-hLC50: 523 mg/L

48-h LC100: 696 mg(L

48-hNOEC: < 27.6 mg/L

Comments: Stock solution of 2204 mg/l of the test item was prepared according to procedure D, as

described in theaspAR Guidelines for Toxicity Testing of Substances and Preparations

Used and Discharged Offshore. See pages 10-11 of this report. Test solutions were

prepared from the Water Accommodated Fraction (WAF).

Note: test results obtained when the test item is prepared according to procedure A refer to

the nominal concentrations of the test item as stated. Test results obtained when the test

item is prepared according to procedures B, C or D refer only to Water-Accommodated

Fractions of the test item prepared at the nominal concentrations of the test item stated.

Preferred pH range is between 7.5 and 8.5, which is natural for seawater. The highest test

concentration (2204 mg Dodicor V 5654/1) gave a lower pH-value than the preferred

range.

The 48-h NOEC refers to the highest test concentration in which no mortality was

observed, and 48-h LCloorefers to the lowest test concentration that gave 100% mortality. These are not statistically determined values.

The test results presented in this report were obtained from tests performed on a sample of

the test item sent to the test facility of Clariant Oil Services, formulated according to the

specifications supplied by the manufacturer or supplier at the time of testing. The test

results cannot be assumed to be valid for samples resulting from subsequent modifications

to the original specifications as supplied.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2020-08-28 to 2020-09-03, with the definitive exposure phase from 2020-08-31 to 2020-09-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ISO 14669: 1999(E)

Version / remarks:

1999

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The samples were analysed with an LC-MS/MS method, implemented under non-GLP and documented finally in the GLP raw data.

Sampling for the analytical monitoring
All WAFs and the control were analytically verified via LC-MS/MS in the fresh media at the start of exposure and at the renewal of the test solutions (0 and 24 hours) as well as in the 24-hours old media at the renewal and at the end of the exposure (24 and 48 hours).
At the start of the exposure and at the renewal of the test solutions (0 and 24 hours), samples were taken after preparation of the WAFs and analyzed.
At the renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken from the test vessels.

Vehicle:

no

Details on test solutions:

Water Accommodated Fraction
Water accommodated fractions (WAF) were prepared because the test item is a UVCB substance with compounds of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2018) and ASTM D6081 (2014). Using this approach, aqueous media will be prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

Preparation of the water accommodated fractions
Six water accommodated fractions (WAF) were prepared with nominal loading rates of the test item in the range of 0.158 to 50.0 mg/L set up in a geometric series with a factor of √10: 0.158 – 0.500 – 1.58 – 5.00 – 15.8 – 50.0 mg/L.
The water accommodated fractions (WAF) 1.58 – 5.00 – 15.8 – 50.0 mg/L were prepared in a tightly closed glass flask with a side-arm near the bottom 3 days prior to the start of the exposure as well as 3 days prior to the renewal of the test solutions. For each loading rate, an appropriate amount of the test item was pipetted onto the surface of an appropriate amount of the dilution water. These dispersions were stirred for 72 ± 2 hours at approximately 100 rpm (preferred vortex depth max 10% of liquid depth by visual inspection) at room temperature. After completion of stirring, the dispersions were allowed to stand for 1 hour at room temperature. Then, the WAFs were removed via the side-arm of the flask (from the approximate bottom of the glass flask). The WAFs were checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. The resulting water accommodated fractions (WAF) were used in the test. Since volumes lower than 1 µL of the test item could not be pipetted, the two lowest WAFs 0.158 and 0.500 mg/L were prepared by dilution of the WAF 1.58 mg/L with dilution water.

Test loading
Per definition of the WAF all terms related to concentration level are given as loading rates because partly dissolved compounds and mixtures cannot be related to concentrations.

Control
Dilution water without test item incubated under the same conditions as the test groups. The control solution was prepared without test item following the same method as specified for the WAFs.

Test organisms (species):

other aquatic crustacea: Acartia tonsa

Details on test organisms:

Test system:
Acartia tonsa DANA

Reason for the selection of the test system
Acartia tonsa is one of the recommended species of the test guideline.

Origin
GUERNSEY SEA FARMS LTD., Parc Lane, Vale, Guernsey C.I. GY3 5EQ

Holding
In glass flasks (> 2 L capacity) with approximately 2 L holding water, at 20  3 °C, 16 hours illumination; light intensity of max. 2200 lx at the water surface, dissolved oxygen concentration above 80 % air saturation, pH 8.0 ± 0.3

Holding water
A commercially available formulation of synthetic seawater (SIGMA Sea Salts) with a salinity of 32 ± 2 S will be used.

Test type:

semi-static

Water media type:

saltwater

Remarks:

synthetic seawater (SIGMA Sea Salts)

Limit test:

no

Total exposure duration:

48 h

Hardness:

see any other information on materials and methods incl. tables

Test temperature:

see any other information on materials and methods incl. tables

pH:

see any other information on materials and methods incl. tables

Dissolved oxygen:

see any other information on materials and methods incl. tables

Salinity:

see any other information on materials and methods incl. tables

Conductivity:

see any other information on materials and methods incl. tables

Nominal and measured concentrations:

0.158 – 0.500 – 1.58 – 5.00 – 15.8 – 50.0 mg/L.

Details on test conditions:

Test method
The study was performed under semi-static conditions with a renewal of the test solutions after 24 hours.

Test duration
48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume
25 mL

Dilution water
Same composition as the holding water

Number of copepods and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each concentration level and the control.

Age of the copepods at the start of the exposure
Large copepodids (Stage 5) or adults were used for the test. The copepods were 17 – 20 days old at the start of the exposure period.
 
Acclimatization
Acclimatization was not necessary, because the composition of the dilution water was equivalent to the holding water.

Application
25 g test solution per replicate were weighed out into each test vessel. This corresponds to 25 mL per test vessel. The copepods were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette.

Test temperature (target)
Nominal range: 20 ± 2 °C

Illumination (target)
Diffuse light

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The copepods were not fed during the study.

Reference substance (positive control):

yes

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

not determinable

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

19.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 3.46 - >50.0

Results with reference substance (positive control):

The percentage of mortality for the reference item 3,5-Dichlorphenole (SIGMA-ALDRICH, batch number MKBZ0947V, purity 100.0%, CAS RN 591-35-5) was determined after 48 hours of exposure from 2020-09-02 to 2020-09-04.

LC50-Value (with Confidence Limits) of the Reference Item 3,5-Dichlorphenole
based on nominal concentrations mg/L, (0 - 48 hours)
Current Study Valid Range
LC50 0.5 mg/L
0.5 - 1.5 mg/L, acc. to ISO 14669 : 1999(E)
Confidence limits 0.3 - 0.5 mg/L

Reported statistics and error estimates:

Methods of evaluation
The LL50-value after 24 hours was estimated empirically from the observed mortality.
The LL50-values given are based on the nominal loading rates of the test item, since the test item is a UVCB substance. Per definition of the WAF, all terms related to the concentration have to be given as loading rate because partly dissolved compounds and mixtures cannot be related to concentrations.


LL50-value and statistical analyses
The LL50-value (after 48 hours) was calculated by sigmoidal dose-response regression with the software GraphPad Prism. The respective 95% confidence limits were calculated from the standard error and the t distribution wherever possible. In the case that the confidence limits could not be calculated by the software, the respective confidence limits were empirically derived from the observation data. All calculations were carried out from the best-fit values with the software GraphPad Prism. The loading rate-effect relationship for mortality after 48 hours is shown graphically.


Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Percentage of Mortality after 24 and 48 hours of Exposure

      (n = 20, divided into 4 replicates with 5 copepods each)

Nominal loading rate of the test item in the WAF

[mg/L]       MORTALITY [%]

      24 hours       48 hours

      Replicates       Replicates

      1       2       3       4       MV       1       2       3       4       MV

50.0       0       20       0       0       5       40       60       40       80       55

15.8       0       0       20       0       5       40       40       20       60       40

5.00       0       0       0       0       0       60       40       20       60       45

1.58       0       0       0       0       0       40       40       40       20       35

0.500       0       0       20       0       5       40       40       60       20       40

0.158       0       0       0       0       0       0       20       0       0       5

Control       0       0       20       20       10       0       0       20       20       10

Absolute Numbers of dead Copepods after 24 and 48 hours of Exposure

      (n = 20, divided into 4 replicates with 5 copepods each)

Nominal loading rate of the test item in the WAF

[mg/L]       NUMBER OF IMMOBILE DAPHNIDS / TOTAL NUMBER OF DAPHNIDS

      24 hours       48 hours

      Replicates       Replicates

      1       2       3       4       Sum       1       2       3       4       Sum

50.0       0 / 5       1 / 5       0 / 5       0 / 5       1 / 20       2 / 5       3 / 5       2 / 5       4 / 5       11 / 20

15.8       0 / 5       0 / 5       1 / 5       0 / 5       1 / 20       2 / 5       2 / 5       1 / 5       3 / 5       8 / 20

5.00       0 / 5       0 / 5       0 / 5       0 / 5       0 / 20       3 / 5       2 / 5       1 / 5       3 / 5       9 / 20

1.58       0 / 5       0 / 5       0 / 5       0 / 5       0 / 20       2 / 5       2 / 5       2 / 5       1 / 5       7 / 20

0.500       0 / 5       0 / 5       1 / 5       0 / 5       1 / 20       2 / 5       2 / 5       3 / 5       1 / 5       8 / 20

0.158       0 / 5       0 / 5       0 / 5       0 / 5       0 / 20       0 / 5       1 / 5       0 / 5       0 / 5       1 / 20

Control       0 / 5       0 / 5       1 / 5       1 / 5       2 / 20       0 / 5       0 / 5       1 / 5       1 / 5       2 / 20

The loading rate-effect relationships for mortality after 48 hours of exposure is illustrated graphically in Figure 1. The LL50-values are based on the nominal loading rates of the test item Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol

Additional Observations during the Definitive Test

All tested loading rates of the test item were colorless and visually clear throughout the exposure period.

Water Quality Parameters

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, salinity, conductivity and water temperature) were within the acceptable limits during the study. During the test period, the temperature in the incubator was 19 – 20 °C.

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal loading rates of the test item Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol, the 48 hours-LL50 for Acartia tonsa DANA was 19.9 mg/L (95% confidence limits: 3.46 – > 50.0 mg/L).

Executive summary:

In the acute lethal toxicity with the marine copepod Acartia tonsa DANA, the effects of the test item Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol (batch number: ESD0033040) were determined at the test facility according to ISO 14669 : 1999(E) from 2020-08-28 to 2020-09-03, with the definitive exposure phase from 2020-08-31 to 2020-09-02.

Since the test item is a UVCB substance with compounds of different water solubility and due to the low aqueous solubility of the test item, the test item solutions were prepared as water accommodated fractions (WAFs). For details, see section 4.2. This procedure is in accordance with the OECD guidance document No. 23 (2018) and ASTM D6081 (2014). Per definition, the exposure has to be expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading level) irrespective of the actual concentration of the test item in the WAF because partly dissolved compounds and mixtures cannot be related to concentrations.

The study was conducted under semi-static conditions over a period of 48 hours with six water accommodated fractions of the test item with nominal loading rates of 0.158 to 50.0 mg/L in a geometric series with a separation factor of √10. Twenty copepods (divided into 4 replicates with 5 copepods each) were exposed to each loading rate and the control.

The concentrations of the test item Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol were analytically verified via LC-MS/MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) as well as in the 24-hours old media at the renewal and at the end of the test (24 and 48 hours) in all test item loading rates and the control.

The measured concentrations of the test item Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol in the fresh media were in the range of < LOQ to 0.290 mg/L at the start of the exposure (0 hours) and at the renewal (24 hours). The measured concentrations of the test item in the old media at the renewal and at the end of the test (24 and 48 hours) were in the range of 0 to 16% of the initially measured values.

EL50-Values (with Confidence Limits)

      (based on the nominal loading rates of the test item)

Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol

Effect values       Test duration

[hours]       Nominal loading rate of the test item

[mg/L]

EL50

(with 95% confidence limits)       24       > 50.0 (95% Cl: Not applicable)

      48       19.9 (95% Cl: 3.46 - > 50.0)

 

Description of key information

Two studies according ISO 14669 was performed with Acartia tonsa was performed. Based on the nominal loading rates of the test item Reaction products of DL-methionine and C18 unsaturated fatty acid chloride and isopropanol, the 48 hours-LL50 for Acartia tonsa DANA was 19.9 mg/L (95% confidence limits: 3.46 – > 50.0 mg/L) in the keay study.

In the supporting study an acute lethality test was performed with Acartia tansa (Dana) using Dodicor V 5654 at nominal

concentrations of 27.6- 2204 mg rl. The following results were obtained:

48-h LC50 : 354 mg/L

24-h LC50: 523 mg/L

48-h LC100: 696 mg(L

48-hNOEC: < 27.6 mg/L

Key value for chemical safety assessment

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

EC50

Effect concentration:

19.9 mg/L