Dibenzylbenzene, ar-methyl derivative

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:

key study

Reliability:

1 (reliable without restriction)

Principles of method if other than guideline:

Paris Commission Guidelines Regarding Harmonisation of Procedures of Approval, Evaluation, and Testing of Offshore Chemicals and Drilling Muds (1990).

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Duplicate aliquots were removed from stock solutions at the commencement of each test and from the contents of each tank at the end of each test, for determination of test material concentration by analysis of total radioactivity.

Vehicle:

no

Details on test solutions:

For the range finding and definitive tests, stock suspensions were prepared by adding 1 litre of filtered seawater to 45.4 µg [14C]-DBT and subjecting the mixture to ultrasonication (20 min) followed by continuous stirring (18-24 h). The suspension was centrifuged (1000 r.p.m., 30 min) using a Jouan model C412 bench centrifuge and total radioactivity measured in the supernatant fraction which was used for testing.

For the definitive test, the supernatant fraction was diluted with filtered seawater to produce the following measured concentrations:

20.6, 9.0, 4.5, 2.3 and 1.1 µg equiv.l-', together with a control (0 µg equiv.l-')

Four tanks were prepared at each concentration, each containing 50 ml of test solution. Five Acartia were added to each tank.

Test organisms (species):

other aquatic crustacea: Acartia tonsa

Details on test organisms:

Acartia cultures were maintained in 10 litre glass tanks containing 10 µm filtered natural seawater. The seawater was replaced continuously using a flow-through system. Acartia cultures were fed on a diet of Rhodomonas sp and Skeletonema costatum. Cultures were fed continuously until the day of study commencement and were not fed during the test.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

no

Test temperature:

21 ± 0.5 °C measured

pH:

7.9-8.0 at test start and 8.1 at end

Dissolved oxygen:

> 96% saturation at start, > 91% at end

Salinity:

Filtered (10 µm) natural seawater was used for maintenance of cultures and as the test medium. The seawater was collected from a site on the East coast of Scotland and was free from known sources of pollution. The seawater typically has a salinity of 32 ± 2% and pH of 8.0 ±0.3.

Nominal and measured concentrations:

measured concentrations: 20.6, 9.0, 4.5, 2.3 and 1.1 µg equiv.l-', together with a control (0 µg equiv.l-')

Details on test conditions:

Culture and test tanks were maintained within the laboratory at a temperature of 20 ± 2°C. A light cycle of 16 h light and 8 h dark applied over the study period, illumination being provided by artificial daylight fluorescent tubes.

Reference substance (positive control):

yes

Remarks:

3.5 dichlorophenol

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

9 µg/L

Basis for effect:

mortality

Remarks on result:

other: ANOVA

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 20.6 µg/L

Basis for effect:

mortality

Details on results:

number of dead Acartia were recorded at 24 h and 48 h.
An Acartia was considered dead if no movement was observed within 10 s following gentle agitation of the test tanks.

Results with reference substance (positive control):

In accordance with UK MAFF guidelines (1993) a reference toxicant (3,5-Dichlorophenol at 1.0 mg/L) was tested during the definitive test. Four tanks containing reference toxicant and 4-5 Acartia were included in the test. The following mortalities were recorded:
Time Cumulative Number of
(h) Mortalities (%)
24 58
48 63
As the percentage mortality after 48 h exposure falls within the acceptance criteria of 20-80% mortalities as specified by UK MAFF, the test is considered to have been valid.

The maximum solubility achieved was 20.6 µg equiv/L. This decreased to 3.9 µg equiv/L alter 48 h also suggesting that the chemical became bound to the test tanks during the test. Similar results were found at the other treatment concentrations and no radioactivity was detected in control tanks throughout the test.

At the highest concentration tested, 15% and 40% mortalities were observed after 24 h and 48 h respectively. After 48 h, 10% mortalities were recorded in the control tanks, between 5% and 10% mortalities were recorded at initial concentrations of between 1.1 and 4.5 µg equiv/L and 15% mortalities at 9.0 µg equiv/L.

Validity criteria fulfilled:

yes

Remarks:

result on reference substance

Conclusions:

At 24 h, the No Observed Effect Concentration (NOEC) was at least 20.6 µg equiv/L, the optimum solubility. At 48 h, the NOEC was 9.0 µg equiv/L and the median lethal concentration (LC50) was greater than 20.6 µg equiv/L

Executive summary:

Acute toxicity study of Dibenzylbenzene, ar-methyl derivativeto wards marine zooplanction (Acartia tonsa) was carried out according to OSPAR Convention draft protocol (now ISO 14669). Several attemps were made for reaching stable concentrations. Maximum achieved concetration in sea water was 20.6 µg/L. Median lethal concentration LC50 was not reached for a exposure period of 48 h (lethality at this measured initial concentration was 40%). NOEC was 9 µg/L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Principles of method if other than guideline:

United Kingdom proposal to ISO/TC/147/SC5/WG2: Water quality - Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea)

GLP compliance:

yes

Remarks:

lab accredited by Norwegian Authority

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

For each test concentration a separate stock solution was prepared, by dissolving the test substance in acetone so that adding 100 µl of stock solution pr. litre of seawater would give the specified test concentration. The concentration of acetone in the controls was 100 µl/l
When preparing test solutions some test substance could visually be observed floating on the water surface at concentrations of 0,5 mg/1 and above, indicating that the solubility in seawater was exceeded.

Test organisms (species):

other aquatic crustacea: Acartia tonsa

Details on test organisms:

The test organisms were supplied as eggs, and were cultivated in accordance with the "Paris Commission - Ring Test Protocol" (VKI). Large copepodites and adults were used in the test.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

no

Hardness:

no data

Test temperature:

20 ± 1 °C

pH:

8.2 at test start and 8.3 at end

Dissolved oxygen:

6.6 mg/L

Salinity:

Natural seawater (salinity: 34 %o) from an unpolluted site in Byfjord was used as basic for the dilution water. The water was filtered through a GF/C filter prior to use.

Nominal and measured concentrations:

Nominal :
0,011 mg/I
0,027 mg/I
0,050 mg/I
0,10 mg/I
0,20 mg/I
0,49 mg/I
1,00 mg/I
1,99 mg/I

Details on test conditions:

The test was run in glass containers (28 ml rolled rim vials) each containing 25 ml of test solution and 5 animais, 4 replicates for each concentration. The vials were kept under dim continuous light (1500 - 2000 lux) at a temperature of 20 ± 1 °C. pH and oxygen measurements were performed as indicated on the data sheet.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.027 mg/L

Remarks on result:

other: ANOVA

Key result

Duration:

48 h

Dose descriptor:

other: LC90

Effect conc.:

0.62 mg/L

Remarks on result:

other: Probit

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.087 mg/L

Remarks on result:

other: Probit 95% CL: 0,054 - 0,132

Key result

Duration:

48 h

Dose descriptor:

LC10

Effect conc.:

0.012 mg/L

Remarks on result:

other: Probit

Details on results:

based on lethality

Results with reference substance (positive control):

LC50-48h = 18 mg/L

Validity criteria fulfilled:

yes

Conclusions:

LC50-48h = 0.087 mg/L based on nominal concentrations

Executive summary:

Acute toxicity towards marine copepods was assessed according to a draft ISO standard. Lethality is the end-point. There is uncertainty around reliability arises from the use of solvent and testing above solubility. No analysis of exposure concentration was carried out. The LC50 being in the range of solubility can be used as a supporting study.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: according to guideline, GLP, labelled test substance measured

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

1ml test solution is transferred by pipetting to LSC vials, exact quantity checked by weighting, 10 ml of Ultimagold Packard scintillation liquid is added, and counting is done without intermediate storage

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 14C-DBT is available in isopropanol solution. 16,5 µl (30 µg DBT) is introduced in 1L test medium and stirred vigorously during 48h, settled, then transferred in tests flasks
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): isopropanol
- Concentration of vehicle in test medium (stock solution and final test solution): 16,5 µl/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: "clone 5" (also known as "clone A")
- Source: IRCHA
- Age at study initiation (mean and range, SD): < 24h
- Method of breeding: in lab, in Elendt M7 medium, fed with Raphidocelis subcapitata
- Feeding during test: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

no

Hardness:

test medium according to ISO 6341
Ca+Mg ions = 2.5 mmol/L

Test temperature:

18-20°C

pH:

7.5 in limit test solution, 7.76 in blank medium

Dissolved oxygen:

7.7 mgO2/L in limit test solution, 8.3 in blank medium

Salinity:

test medium according to ISO 6341

Nominal and measured concentrations:

Limit (saturated) test initial measured concentration : 0.029 mg DBT/L
Limit (saturated) test final measured concentration : 0.027 mg DBT/L
Final/initial = 93%

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: penicillin type 120 ml, closed with sealed PTFE septa, fill volume : 120 mL (no headspace)
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ultra-pure water
- Ca/mg ratio: 4
Components:
11.76 g CaCl2, 2H2O/L
4.93 g Mg SO4, 7H2O/L
2.59 g ofNaHCO3/L
0.23 g of KCl/L

- Light intensity: protected from light


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
mobility

TEST CONCENTRATIONS
Saturated solution (measured value = 0.029 - 0.027 mg/L)
- Justification for using less concentrations than requested by guideline: poor solubility: limit test at WSL

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 0.029 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: water solubility limit in test conditions measured through 14C

Key result

Duration:

48 h

Dose descriptor:

LC0

Effect conc.:

>= 0.029 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: water solubility limit in test conditions measured through 14C

Details on results:

QUALITY CRITERIA:
- Control mortality (OK if < 10%): OK
- Daphnia in control were not trapped at the surface of the water: no
- pH (OK if equals to initial pH ± 1): OK
- Dissolved oxygen (OK if > 2mg/L): OK
- Test substance concentration (OK if > 80% of initial concentration): OK

Validity criteria fulfilled:

yes

Remarks:

Control mortality (OK if < 10%): OK - pH (OK if equals to initial pH ± 1): OK - Dissolved oxygen (OK if > 2mg/L): OK - Test substance concentration (OK if > 80% of initial concentration

Conclusions:

No immobilisation of daphnia was observed for exposure during 48 h at solubility limit in test medium. Therefore EC50 of Dibenzylbenzene, ar-methyl derivative at 48 h for Daphnia magna was determined to be > 0.029 mg/L, found to correspond to the solubility limit of the tested substance in the mineral medium used for this test.

Executive summary:

The acute toxicity (inhibition of mobility) of Dibenzylbenzene, ar-methyl derivative for Daphnia magna was assessed according to the method C2 of the European Directive 92/69/CEE (equivalent to OECD 202). Daphnia were exposed in a static test to a concentration of about 0,029 mg/l. The test was performed with 5 daphnia per vessel. The test was performed using closed flasks entirely filled with test solutions and closed with butyl rubber caps covered with PTFE. The test concentrations of Dibenzylbenzene, ar-methyl derivative were measured by Liquid scintillation counting.

No inhibition of mobility was observed after 24 or 48 hours at a tested concentration of about 0,029 mg/L of Dibenzylbenzene, ar-methyl derivative. This concentration is the solubility limit of Dibenzylbenzene, ar-methyl derivative in the mineral medium used for the test.

The method was applied with respect to its quality criteria :

Immobilisation in the control did not exceed 10 % at the end of the test ;

Concentration of dissolved oxygen in the test vessels remained above 2 mg/L at the end of the test and pH did not vary by more than 1 unit ;

The concentrations of the test substance have been maintained to within 80 % of the initial concentration throughout the duration of the test.

Description of key information

No acute EC50 is determined in freshwater and marine water invertebrates up to solubility limit.

Additional information

Four studies are available in fresh water and two in sea water.

Three Daphnia magna studies (Scholz 1990 & 1994, Knacker 1990) are considered as not reliable as they make use of a surfactant to expose the animals at concentrations well above solubility limit. In addition the surfactant concentration varies with the test substance concentration which does not allow to separate effects of the two. In the fourth Daphnia test (Thiébaud, 1996), no immobilization was observed after 48h exposure at the highest soluble (measured) concentration, 29 µg/L.

In the first Acartia test (Rogaland 1996) a LC50 -48h has been determined as being 87 µg/L (nominal), using acetone as solvent.

In the second Acartia test (Inveresk, 1997) LC50 was not attained at the highest soluble concentration of 20.6 µg/L.

It can be concluded that in normal conditions (not trespassing water solubility limit) there is no significant acute toxicity towards fresh- or seawater invertebrates.