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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25/06/2002 - 05/09/2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Valid and conclusive guideline study under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
as at 1984
Deviations:
yes
Remarks:
mobility and mortality only assessed after 48 h
GLP compliance:
yes
Remarks:
- according to OECD GLP Principles of Good Laboratory Practices (1998)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all concentrations before start of experiment and at the end
- Sampling method: On day 0 and 2 samples of 0.5 mL were taken in duplicate (one sample served as contra sample and was stored at a temperature of 2 - 8°C) from all test flasks.
- Sample storage conditions before analysis: refrigerated (2 - 8 °C)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- On day before test initiation a stock solution with a concentration of 160 mg TCP per litre Elendt M4 medium was prepared in a glass flask by dissolving 130 µL (= 180 mg) TCP in 1120 mL Elendt M4 medium. The glass flask was closed with a screw cap with teflon inlay and placed in the dark at a temperature that ranged between 19.8 and 20.8°C. The stock solution was stirred continuously.
Per concentration 3 flasks of 320 mL were filled with an amount of the stock solution and Elendt M4 medium according to Table 1. The flasks were closed with a screw cap with teflon inlay. An extra test flask with a concentration of 40 mg TCP was prepared to measure pH, temperature and oxygen content at test initiation. Three control flasks were prepared.
- Dilution: 0, 5, 10, 20, 40 or 80 mL of stock solution were adjusted each with the respective volume of Elendt M4 medium to 320 mL to give final solutions of 0, 2.5, 5.0, 10, 20 and 40 mg/L of test item.
- Chemical name of vehicle: no vehicle
- Evidence of undissolved material: all test solutions were clear, no undissolved material
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain: Daphnia magna
- Source: RIZA, Lelystad, The Netherlands
- Age at study initiation: 0 - 24 h
- Method of breeding: Water fleas of the same age are cultured in vessels with 2 litre Elendt M4 medium. They are fed with an algal suspension of Chlorella vulgaris. The water fleas were in culture at the laboratory of the Health Environment & Safety Department of Solvay Pharmaceuticals since 9 April 2002. The water fleas are cultured in a climate chamber with a temperature set point of 20.0 °C. The light regime is 16 hours light and 8 hours darkness per day. The light is switched on at 6 a.m. Each week a new culture of water fleas is started and water fleas older than 3 weeks are removed.
- Feeding during test: no feeding, 48 h test

ACCLIMATION
- Acclimation period: not needed, animals are breeded in the test medium (Elendt M4 medium)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
Not explicitly stated in the report, but may be derived from the test media composition, see table 1 in “any other information on materials and methods including tables”
Test temperature:
20.9 to 21.2 °C, see table 2 in “any other information on materials and methods including tables” for details
pH:
8.0, see table 2 in “any other information on materials and methods including tables” for details
Dissolved oxygen:
At 0 hours, dissolved oxygen was 8.9 mg/L in Elendt M4 medium and the highest concentration. At 48 hours, dissolved oxygen ranged between 8.5 and 8.7 mg/L. Dissolved oxygen saturation at 20 °C is 9.2 mg/L.
see table 2 in "any other informations on materials and methods including tables"
Salinity:
Not explicitly stated in the report, but may be derived from the test media composition, see table 1 in “any other information on materials and methods including tables”
Nominal and measured concentrations:
Nominal: 0, 2.5, 5.0, 10, 20, 40 mg/L
Measured: n.d. (not detected), 2.5, 4.7, 8.4, 15, 27 mg/L
See table 3 in "any other informations on materials and methods including tables"
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed
- Material, size, headspace, fill volume: closed glass flasks with a volume of 320 mL, completely filled, no headscpace
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: prepared M4 medium, see table 1 in "any other informations on materials and methods including tables"
- Culture medium different from test medium: no
- Intervals of water quality measurement: at start and end of test

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness per day
- Light intensity: not reported

EFFECT PARAMETERS MEASURED:
mobility, including partial immobility; observed only at end of test as the test vessels were not transparent and therefore an observation after 24 h of incubation was precluded

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
no
Remarks:
but historic positive controls added in the protocol (see below)
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
15 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
8.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: partial immobility, sublethal effects
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
8.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: partial immobility, sublethal effects
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
>= 15 - <= 27 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks:
- Extrapolated value, no concentration gave partial immobilisation
Remarks on result:
other: 95% confidence interval
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
ca. 20 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks:
- Extrapolated value, no concentration gave partial immobilisation
Details on results:
- Behavioural abnormalities: no
- Mortality of control: No immobility was observed in the control glass beakers
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

No immobility was observed in the control glass beakers and therefore the test meets the validity criterion of ≤ 10 % control immobility. The NOEC based on immobility and mean measured concentrations was equal to 15 mg/L. The NOEC based on sublethal effects was 8.4 mg/L. The EC50 48h based on mean measured concentrations was 20 mg/L. The 95 % confidence interval of the EC50 48h was 15-27 mg/L.

The endpoints of the test can also be based on geometric mean measured concentrations. In this case the NOEC based on immobility and geometric mean measured concentrations was equal to 14 mg/L. The NOEC based on sublethal effects was 8.3 mg/L. The EC50 48h based on geometric mean measured concentrations was 19 mg/L. The 95 % confidence interval of the EC50 48h was 14-27 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? Yes, see above
Reported statistics and error estimates:
The No Observed Effect Concentration (NOEC) based an immobility and mean measured concentrations was determined with Fisher's Exact Test (Sokal & Rohlf 1981).
As there was no concentration with partial immobility, the geometric mean of the highest concentration without immobility and the lowest concentration with 100 % immobility was regarded as the EC50 48h.

- Table 4: Number of mobile daphnids during the test

Mean measured
concentration

[mg/L]

Number of mobile daphnidsA

Mean immobility at
test termination

[%]

0 hoursB

48 hours

0

10

10

 

 

10

10

0

 

10

10

 

2.5

11

11

 

 

10

10

0

 

10

10

 

4.7

10

10

 

 

10

10

3.3

 

10

9

 

8.4

10

10

 

 

10

10

0

 

10

10

 

15

10

10 (10)

 

 

10

10 (10)

0

 

9

9 (9)

 

27

10

0

 

 

11

0

100

 

10

0

 

A Between brackets the number of mobile daphnids showing sublethal effects is given. Sublethal effect in this study was less activity.

B Not observed an day 0 but extrapolated from the observations after 48 hours.

Validity criteria fulfilled:
yes
Conclusions:
The test compound was tested for acute toxicity to water fleas according to OECD TG 202.
The NOEC based on immobility was found to be 15 mg/L.
The NOEC based on sublethal effects (partial immobility) was found to be 8.4 mg/L
The EC50-48h based on immobility was found to be 20 mg/L (15 - 27 mg/L 95%CL)
Executive summary:

The acute toxicity of 1,2,3-trichloropropane to water fleas (Daphnia magna) was tested following OECD Guideline 202 (1984) and according to OECD GLP Principles of Good Laboratory Practice and Compliance Monitoring No. 1, ENV/MC/CHEM/(98)17 of 21 January 1998.

The test was conducted at nominal concentrations of 0, 2.5, 5.0, 10, 20 and 40 mg/L. Because 1,2,3-trichloropropane is volatile, the test was conducted in closed glass flasks with a volume of 320 mL. Three flasks, each with 320 mL test solution, were prepared per concentration and 10 daphnids were added to each flask. The experiment was terminated alter 48 hours of exposure.

To study the exposure of the daphnids to 1,2,3-trichloropropane, samples of the test solutions were taken at the start and at the end of the test and analysed with Gas Chromatography. The difference between the nominal and the mean measured concentrations was larger than 20 % and for this reason the biological endpoints of the test were based on the average of the measured concentration being 0, 2.5, 4.7, 8.4, 15 and 27 mg/L.

No immobility was observed in the control glass beakers.

The No Observed Effect Concentration (NOEC) based on immobility and mean measured concentrations was determined with Fisher's Exact Test (Sokal et al 1981) and was equal to 15 mg/L.

The NOEC based on sublethal effects was 8.4 mg/L.

As there was no concentration with partial immobility, the geometric mean of the highest concentration without immobility (= 15 mg/L) and the lowest concentration with 100 % immobility (= 27 mg/L) can be regarded as the 48-h EC50.

The EC50 based on mean measured concentrations was 20 mg/L.

The 95 % confidence interval of the EC50-48h is 15 - 27 mg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Valid GLP guideline study with significant methodological deficiencies. Actual measurements of test concentrations were not performed. The test item is quite volatile, thus the exposure unclear.
Qualifier:
according to guideline
Guideline:
other: Committee on Methods for Toxicity Tests with Aquatic Organisms, Methods of Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians, 1975, U.S. EPA, Ecol. Res. Ser. 660/3-75009
Qualifier:
according to guideline
Guideline:
other: American Public Health Association, 1980, Standard Methods for the Examination of Water and Wastewater, 15th ed. Washington, DC, 1134 p. (Due to volatilization the actual exposure is unclear, as no analytical dose verification was performed.)
GLP compliance:
yes
Remarks:
- according to OECD 1981, U.S. FDA 1978, and U.S. EPA 1983
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
Test concentrations were prepared based on total compound and were corrected for sample purity. Acetone was used in the preparation of all working stock solutions.
The solvent control received an aliquot 0.020 mL of acetone equivalent to that of the highest test concentration.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: Wildlife daphnids were identified to species using a standard taxonomic key
- Age at study initiation: first instar less than 24-hours old
- Feeding during test: No

ACCLIMATION
- Type and amount of food: The Daphnia were fed algae (Selenastrum capricornutum) at least every three days prior to testing and supplemented with a suspension of fish food.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Hardness (CaCO3) = 225-275 ppm
Test temperature:
20 ± 2 °C during the test
pH:
The pH was 8.1 in control water at 0 hours. At 48 hours, pH was 8.5-8.6.
Dissolved oxygen:
8.1 mg/L in control water at 0 hours
8.7-8.8 mg/L at 48 hours
(Dissolved oxygen saturation at 20 °C is 9.2 mg/L)
Nominal and measured concentrations:
Nominal concentrations tested were:
Solvent control, 0, 5.6, 10, 18, 32, 56 mg/L; these concentrations were a logarithmic series
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 250-ml glass beakers containing 200 mL of the test medium
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well water
- Metals: see Table 1
- Pesticides: see Table 1
- Conductivity: see Table 1
- Culture medium different from test medium: yes, probably since wildlife animals were used

OTHER TEST CONDITIONS
- Photoperiod: 16-hour daylight photoperiod, with 30 minute simulated dawn and dusk periods
- Light intensity: 50-70 foot-candles


EFFECT PARAMETERS MEASURED
Every 24 h for observation of mortality and abnormal effects such as surfacing, clumping of the daphnids together and daphnids lying on the bottom of test chambers.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Mortality after 24 h 0, 1, 10 dead daphnids, after 48 h 0, 2, 10 respectively
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
behaviour
Remarks on result:
other: 95% CL 29-78 mg/L
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
20
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
behaviour
Remarks on result:
other: 95% CL 17-24 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
behaviour
Reported statistics and error estimates:
The 24- and 48-hour LC50 values and corresponding 95-percent confidence limits were determined by an LC50 computer program developed. This program calculated the LC50 statistic and it 95-percent confidence limits using the binomial, Moving Average and Probit methods. The method of calculation selected was that which gave the narrowest confidence limits for each separate analysis. This was the Probit Method for the LC50 24 h and the Moving Average Mathod for the LC50 48 h.

TABLE 2: Mortality Rates and Water Quality Measurements During the Acute Toxicity Test of TCP to Daphnia magna

Nominal Concentration [mg//L]

Percent mortality

Water quality

24 h

48 h

0 h

48 h

°C

Dissolved Oxygen

pH

°C

Dissolved Oxygen

pH

Control

0

0

20

8.1 mg/L

8.1

20

8.7 mg/L

8.5

Solvent control

0

0

 

 

 

 

 

 

5.6

0

0

20

8.7 mg/L

8.5

10

10

20

 

 

 

18

35

50

20

8.7 mg/L

8.5

32

40

55

 

 

 

56

55

100

20

8.8 mg/L

8.6

 

Validity criteria fulfilled:
yes
Conclusions:
The data do not provide sufficient information to support the conclusion regarding the 24- and 48-hour LC50s.
Executive summary:

The acute toxicity of Trichloropropane to Daphnia magna was assessed using the methods outlined by the Committee on Methods for Toxicity Tests with Aquatic organisms. Thus the study was conducted in accordance with a recognized scientific method for determining acute toxicity to daphnia, and in accordance with GLP regulations. Water quality parameters and temperature, dissolved oxygen and pH were measured at the termination of the test and were within acceptable limits.

There was no chemical analysis performed to verify or validate the test concentration. Owing to the demonstrated volatility of 1,2,3-trichloropropane, a 48-hour water evaporation study was performed to determine the actual concentrations to which the daphnia were exposed. These results verified that the test material is a volatile compound for which actual concentrations must be measured throughout the 48-hour study.

All reported values were based upon nominal concentrations. The 48-hour LC50 for the Compound Trichloropropane was found 20 mg/L (95% C.I.). The no-effect level observed for Trichloropropane as 5.6 mg/L after 48 hours, which was based on the lack of mortality and abnormal effects.

The test item will evaporate from the aqueous phase to some extent, thus reducing the exposure concentration for aquatic species (Solvay 2001). For this reason the study is considered not reliable.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only secondary literature available
Reason / purpose for cross-reference:
reference to other study
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
unfed
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Details on test conditions:
unfed
Duration:
48 h
Dose descriptor:
IC50
Effect conc.:
35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
240 µmol/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only secondary literature available
Reason / purpose for cross-reference:
reference to same study
GLP compliance:
no
Analytical monitoring:
not specified
Vehicle:
no
Test organisms (species):
other aquatic crustacea: Chaetogammarus marinus
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
60 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
45 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mortality
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
publication submitted 24 July 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with chemical analysis but without detailed documentation and not GLP, conducted on 11 compounds in parallel
Qualifier:
according to guideline
Guideline:
other: US EPA (1993). Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms, 4th edition Weber CI (ed) EPA report EPA/600/4-90/027F, Washington DC, U.S.A. 293 pp.
Qualifier:
according to guideline
Guideline:
other: ASTM E1192 Standard Guide for Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents with Fishes, Macroinvertebrates, and Amphibians
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,2,3-Trichloropropane
- Substance type: (non-polar narcotic) organic
- Analytical purity: 97 %
- source unknown
Analytical monitoring:
yes
Vehicle:
yes
Details on test solutions:
The stock solution was made using nanograde acetone.
Test organisms (species):
Ceriodaphnia sp.
Details on test organisms:
The cladoceran used in this study was identified as Ceriodaphnia dubia Richard. However, it was not identical to C. dubia (Juni at aI 1990) and to denote this it has been called Ceriodaphnia cf. dubia.
The C. cf dubia were maintained under conditions presented elsewhere (Warne 1996).
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
65.2 mg/L as CaCO3
pH:
7.7 (mean value)
Nominal and measured concentrations:
The loss during the study was less than 20 %.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass rectangular bottles with screw-on teflon-lined lids
- Type: closed
- 214mL, elimination of any headspace in order to minimize volatilization

The test animals were fed during exposition (Kielhorn et al 2003)

EFFECT PARAMETERS MEASURED:
The number of immobilized cladocerans as defined by ASTM (1988) were recorded after 48 h.
Reference substance (positive control):
yes
Remarks:
11 compounds tested in parallel and used for a QSAR development
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
4.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
>= 2.7 - <= 6.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
28 µmol/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
>= 18 - <= 44 µmol/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The EC50 48 h (immobilization) value and 96% confidence limit were based on the initially measured concentration as the loss during the study was less than 20 %.
The EC50 48 h based was 4.1 mg/L (28 μmol/L). The 95 % confidence interval of the EC50 48h was 2.7-6.5 mg/L (18-44 μmol/L).
Reported statistics and error estimates:
The EC50 48h (immobilization) value and 96% confidence limit were determined by trimmed Spearman-Karber method using the initial measured concentrations.
Validity criteria fulfilled:
yes
Conclusions:
As for all 11 non-polar narcotic chemicals tested 1,2,3 Trichloropropane was significantly more toxic to the cladoceran Ceriodaphnia dubia Richard. An EC50 of 4.1 mg/L was measured.
Executive summary:

The toxicity of eleven non-polar narcotic chemicals to the cladoceran Ceriodaphnia cf. dubiawas determined according to ASTM and U.S. EPA standard protocols. The test organisms C. cf. dubiawas with regard to molar concentrations found to be approximately four times more sensitive to all of these narcotic chemicals than Daphnia magna tested under virtually identical conditions by Hermens et al (1984). The toxicity data were also used to develop and validate quantitative structure-activity relationships (QSARs) and thus 10 references were tested with results in line.

Description of key information

Invertebrates represent the most susceptible organism group in the aquatic environment. Although marine species data exist the dose descriptors for both freshwater and marine water are assigned to the lowest value obtained from freshwater, due to reliability considerations .

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
4.1 mg/L

Marine water invertebrates

Marine water invertebrates
Effect concentration:
4.1 mg/L

Additional information

For those invertebrates tested, the lowest 48-h EC50 value of 4.1 mg/L with the endpoint immobilization, which was about 5 times lower than the corresponding value from daphnids, was reported by Rose et al (1998) for a cladoceran (Ceriodaphnia cf. dubia). The EC 50 for daphnids (Daphnia magna) was found to be 20 mg/L (de Groot 2002). Both studies followed a guideline protocol and are considered valid and conclusive.

For marine species a value is available from Kooijman (1981). The test organism employed was (Chaetogammarus marinus) and the measured endpoint was LC50 after 48 h of 45 mg/L. Since the latter is not reliable because of lacking analytical dose verification and supposed vaporisation of TCP during the exposition, the freshwater value should be used in assessment for the marine compartment as well.