Decanoic acid, mixed esters with dipentaerythritol, heptanoic acid and octanoic acid.

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 April 2004 to 20 May 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

not specified

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

All samples were stored frozen. On the day of analysis, the frozen samples were defrosted at room temperature. The entire volume of each sample (5mL) was transferred quantitatively into a 20mL volumetric flask using methanol. The flasks were filled up to the mark with methanol.

Vehicle:

no

Details on test solutions:

The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system were prevented as much as possible (e.g. film of the test substance on the water surface).
Hatcol 5127 is a light yellow liquid. The water so1ubility of Hatcol 5127 at 20.4 +/-7˚C determined to be < ס.14X10^-3 g/l in a preliminary experiment (pH was between 6.0-8.1, NOTOX Project 388035).
AII test solutions with a loading rate at or above 1.0 mg/l were prepared separately (loading rates of 1.0, 10 and 100 mg/l). These saturated solutions were stirred for three days to reach maximum solubility. After the stirring period the 100 mg/l loading rate was slightly hazy and contained a test substance floating layer and a test substance precipitate. The 1.0 and 10 mg/l loading rates were observed to be clear and colourless. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance (see also NOTOX Project 388035). After the stirring period all three mixtures/solutions were therefore filtered twice. First through a glass fiber pre-filter (Schleicher and Schuell GF92) to remove the larger undissolved test substance particles and subsequently through a membrane filter (Schleicher and Schuell RC55, 0.45µm). Finally a ten-fold dilution was prepared from the filtrate prepared at a loading rate of 1.0 mg/l and indicated as 0.1 mg/l. AII test solutions were clear and colourless. Note that the blank-control received the same treatment as the test concentrations.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, adequate volumes of an algal suspension were added to each replicate providing a cell density of 104 cells/ml.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Selenastrum capricornutum, Strain: NIVA CHL 1.
Source: ln-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

CaMg 0.24 mmol (24 mg CaCO3/L)

Test temperature:

23.9 and 24.3°C

pH:

6.0-9.0

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations 1.0, 10.0, 100.0 mg/L

Details on test conditions:

Test vessels: 100ml, all glass containing 50 ml of test medium
Medium: M2
Cell density: An initial density of 1 x 10^4 cells/ml.
Illumination: Continuously using TLD lamps of the type 'Cool-white' of 30 Watt. with a light intensity within the range of 84 to 113µE.m-2.s-1.
Incubation: During incubation the algal cells were kept in suspension by continuous shaking.


Fresh water algae culture
Stock culture: Algae stock cu1tures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate roam at a temperature of 23 +/- 2˚C.
Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; formulated using Milli-Ro water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3: 500 mg/L
K2HPO4: 40 mg/L
MgS04.7H20: 76 mg/L
Na2C03.10H20: 54 mg/L
C6H8O7.H2O: 6 mg/L
NH4N03: 330 mg/L
CaCl2.H20: 36 mg/L
C8H5FeO7.xH20: 6 mg/L
H3BO3: 2.9 mg/L
MnCl2.4H20: 1.81 mg/L
ZnCl2: 0.11 mg/L
CuSO4.5H2O: 0.08 mg/L
(NH4)5Mo7O24.4H2O: 0.018 mg/L

Pre-culture: 3 days before the start of the test, cells from the algal stock culture inoculated in culture medium at a cell density of 2.10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium: M2; according to the ISO-Standard "Algal growth inhibition test” Nov. 1989; formulated using Milli-Q water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Mllli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation and with the following composition:
NH4Cl: 15 mg/L
MgCl2.6H2O: 12mg/L
CaCl2.H20: 18 mg/L
MgS04.7H20: 15mg/L
K2HPO4: 1.6 mg/L
FeCl3.6H2O: 80 µg/L
Na2EDTA.2H2O: 100 µg/L
H3BO3: 185 µg/L
MnCl2.4H20: 415 µg/L
ZnCl2: 3 µg/L
CoCl2.6H2O: 1.5 µg/L
CuCl2.2H2O: 0.01 µg/L
Na2MoO4.2H2O: 7 µg/L
NaHCO3: 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/l (24mg CaCo3/l)
pH 8.3+/-0.2

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass/growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval 4.7-27

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval 50-300

Remarks:

initial concentration Hatcol 5127 (mg/L): > 0.015

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 15-47

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 1500-14000

Remarks:

initial concentration Hatcol 5127 (mg/L): > 0.015

Details on results:

Measured substance concentrations
The concentrations of Hatcol 5127 were determined by Gas Chromatography with mass spectrometric detection (GC-MS). The test substance consisted of a mixture of molecules with different molecular weights, which differed in water solubility, resulting in a number of peaks in the chromatogram of the test substance solutions. It was not possible to determine which molecule was responsible for the toxicological response, if any. Furthermore, since not all components were quantifiable in the calibration chromatograms, it was not possible to determine the concentration of the total test substance. Therefore, the toxicological evaluation was based no the loading rate. ln addition, the actual concentration was estimated from the largest peak observed in the chromatograms of Hatcol 5127.
The analytical results showed that the filtered solution prepared at a loading rate of 100 mg/l contained an initial concentration of 0.015 mg/l. After 24 hours of exposure this test concentration had decreased to below the lowest calibration solution (<0.001 mg/l). The lower test concentrations were below the lowest calibration solution from the start of exposure.

Inhibition of cell growth and reduction of growth rate
No inhibition of cell growth was observed at a loading rate of 0.1 mg/l. Statistically significant inhibition of cell growth was found at loading rates of 1.0 and 100 mg/l (Bonferroni t and Tukey test, a = 0.05). Growth rates were in the range of the controls at the loading rates of 0.1, 1.0 and 10 mg/l during the 72-hour test period. Reduction of growth rate was 20% at a loading rate of 100 mg/l. Statistically significant reduction of growth rate was found at loading rates of 1.0 and 100 mg/l (Bonferroni t and Tukey test, a = 0.05).
Note that the statistically significant cell growth inhibition and growth rate reduction at a loading rate of 1.0 mg/l was biologically not relevant since there was no statistical significance at a loading rate of 10mg/l.

Results with reference substance (positive control):

Under the conditions of the reference study with Selenastrum capricornutum, potassium dichromate inhibited cell growth and reduced growth rate of this fresh water algae species at nominal concentrations of 0.18 and 0.56 mg/l, respectively, and higher.
The EC50 for cell growth inhibition (E8C50: 0-72h) was 0.49 mg/l with a 95% confidence interval ranging from 0.28 to 0.85 mg/l. The historical ranges of the 72h EC50 for growth inhibition lie between 0.49 and 1.4 mg/l. Hence, the EBC50: 0-72h for the present batch corresponds with this range.
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.0 mg/l with a 95% confidence interval ranging from 0.58 to 1.8 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the present batch corresponds with this range.

Mean cell densities (x 10^4 cells/ml) during the final test

Loading rate Hatcol 5127 (mg/L)	Exposure time (hours)
	0	24	48	72
Blank control	1.0	4.8	27.3	103.5
0.1	1.0	5.0	27.8	113.3
1.0	1.0	4.9	24.4	85.1
10	1.0	5.7	25.3	92.3
100	1.0	5.3	20.7	40.1

 

Percent inhibition of cell growth during the final test

Loading rate Hatcol 5127 (mg/L)	Cell growth (0-72 hours)
	Mean area (A)	Inhibition (%)
Blank control	1952.46
0.1	2086.12	-6.8
1.0	1666.24	14.7
10	1793.44	8.1
100	1045.36	46.5

 

Percentage reduction of growth rate at different time intervals during the final test

Loading rate Hatcol 5127 (mg/L)	Mean growth rate
	µ (0-24 hours)	Reduction (%)	µ (0-48 hours)	Reductions (%)	µ (0-72 hours(	Reduction (%)
Blank control	0.06538		0.06889		0.06441
0.1	0.06736	-3.0	0.06923	-0.5	0.06562	-1.9
1.0	0.0665	-1.8	0.06654	3.4	0.06171	4.2
10	0.07257	-11.0	0.06734	2.2	0.06281	2.5
100	0.06968	-6.6	0.06308	8.4	0.05127	20.4

 

pH levels recorded during the final test

Loading rate Hatcol 5127 (mg/L)	Exposure time (hours)
	0	72
Blank control	7.7	9.6
0.1	7.6	9.0
1.0	7.7	8.9
10	7.5	8.8
100	6.4	8.2

 

Individual cell densities

Number of inoculated cells at t=0 1 x 10

Loading rate Hatcol 5127 (mg/L)	Vessel number	Exposure time (hours)
		0	24	48	72
Blank control	1 2 3 4 5 6	1.0 1.0 1.0 1.0 1.0 1.0	4.95 5.04 4.58 4.66 4.72 4.88	26.90 27.42 27.26 26.14 26.51 29.69	107.84 91.14 103.84 96.73 106.57 111.61
0.1	1 2 3	1.0 1.0 1.0	5.06 4.92 5.13	27.60 27.18 28.48	112.10 99.61 128.08
1.0	1 2 3	1.0 1.0 1.0	4.91 4.65 5.27	26.30 22.82 24.16	90.45 81.34 83.55
10	1 2 3	1.0 1.0 1.0	5.43 6.28 5.45	25.19 25.83 25.02	98.70 96.09 82.17
100	1 2 3	1.0 1.0 1.0	5.01 5.58 5.40	19.38 21.62 21.01	39.33 40.43 40.58

 

 Calculation of growth (area under the growth curve) and growth rate

Loading rate Hatcol 5127 (mg/L)	Vessel number	Area (A)	Growth rate			Growth inhibition (%)	Growth rate reduction (%)
		0-72hrs	0-24hrs	0-48hrs	0-72hrs	0-72hrs	0-24hrs	0-48hrs	0-72hrs
Blank control	1 2 3 4 5 6	1998.48 1848.72 1950.24 1839.96 1968.36 2109.00	0.06664 0.06739 0.06340 0.06413 0.06466 00.6605	0.06859 0.06898 0.06886 0.06799 0.06828 0.07064	0.06501 0.06312 0.06448 0.06350 0.06484 0.06549	1952	0.06538	0.06889	0.06441
0.1	1 2 3	2069.04 1905.72 2283.60	0.06756 0.06404 0.06925	0.06912 0.06880 0.06978	0.06555 0.06391 0.06740	-6 2 -17	-3 -2 -4	-2 1 -5
1.0	1 2 3	1774.44 1575.35 1648.92	0.06630 0.06404 0.06925	0.06812 0.06516 0.06635	0.06257 0.06109 0.06146	9 19 16	-1 2 -6	3 5 5
10	1 2 3	1859.28 1863.72 1657.32	0.07050 0.07656 0.07065	0.06722 0.06774 0.06708	0.06378 0.06341 0.06123	5 5 15	-8 -17 -8	1 2 5
100	1 2 3	997.32 1077.96 1060.80	0.06714 0.07163 0.07027	0.06176 0.06403 0.06344	0.05100 0.05138 0.05143	49 45 46	-3 -10 -7	21 20 20

 

Overview of % inhibition in cell growth and % reduction growth rate in the reference test:

Concentration K2Cr2O7 (mg/l)	Total cell growth:		Growth rate:
	Mean area (0-72hr)	Inhibition (%)	Interval (0-72hr)	Reduction (%)
Blank control	3665.52		0.07367
0.18	3041.24	17.0	0.07077	3.9
0.32	2737.76	25.3	0.06957	5.6
0.56	1744.52	52.4	0.06262	15.0
1.0	524.76	85.7	0.03993	45.8
1.8	234.40	93.6	0.01361	81.5
3.2	157.48	95.7	0.00664	91.0

 

 

Validity criteria fulfilled:

yes

Conclusions:

Due to the fact that test concentrations below or approximating the water solubility level of Hatcot 5127 could not be analysed as these were below the limit of quantification, a different approach was followed for the calculation of EC and NOEC-values.

To this respect the following text was quoted from the OECD guidance on classification of chemicals is applicable: ‘Many substances covered by the classification scheme are in fact mixtures, for which measurement of exposure concentration is difficult, and in some cases impossible. Substances such as petroleum distillate fractions, polymer, substances with significant levels of impurities, etc can pose special problems since the toxic concentration is difficult to define and impossible to verify. Typical testing procedures often rely on the formation of a Water Soluble Fraction (WSF) or Water Accommodated Fraction (WAF) and data are reported in terms of loading rates. These data may be used in applying the classification criteria. Organisation for Economic Co-operation and Development (OECD), OECD guidance document on the use of the harmonised system for the classification of chemicals which are hazardous for the aquatic environment, OECD series on testing and assessment number 27, July 23 2001)

Under the conditions of the present study with Selenastrum capricornutum, Hatcol 5127 reduced growth rate of this fresh water algae species significantly at a loading rate of 100 mg/l (both statistical and biological).
The EL50 for cell growth inhibition (EBL50: 0-72h) was beyond the range tested.
The EL10 for cell growth inhibition (EBL10: 0-72h) was 11 mg/l with a 95 % confidence interval ranging from 4.7 to 27 mg/l.
As growth rate is derived from the slope under the growth curve in a logarithmic plot, the measure of the specific growth rate is preferable over biomass following from the mathematical nature of exponential growth.
The EL50 for growth rate reduction (ERL50: 0-72h) was beyond the range tested.
The EL10 for growth rate reduction (ERL10: 0-72h) was 26 mg/l with a 95 % confidence interval
ranging from 15 to 47 mg/l.
The NOELR for both cell growth inhibition and growth rate reduction was 10 mg/l.

Executive summary:

Selenastrum capricornutum, fresh water algal growth inhibition test with Hatcol 5127.

The study procedures described in this report were based on the ISO international standard 8692 1989. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.3, 1992, the OECD guideline No 201 1984 and the OECD series on testing and assessment number 23 December 14 2000.

Hatcol 5127 is a light yellow liquid. The water solubility of Hatcol 5127 at 20.4 +/- 0.7˚C was determined to be < 0.14 x 10-3 g/l in a preliminary experiment (pH was between 8.0-8.1, NOTOX Project 388035).

 

All test solutions with a loading rate at or above 1.0 mg/l were prepared separately (loading rates of 1.0, 10 and 100 mg/l). These saturated solutions were stirred for three days to reach maximum solubility. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance (see also NOTOX Project 388035). After the stiring period all three mixtures/solutions were therefore filtered twice. First through a glass fiber pre-filter to remove the larger undissolved test substance particles and subsequently through a membrane filter (0.45µm). Finally a ten-fold dilution was prepared from the filtrate prepared at a loading rate of 1.0 mg/l. All test solutions were clear and colourless.

A final test was performed exposing exponentially growing algae to 0.45µm filtered solutions prepared at loading rates of 1.0, 10 and 100 mg/l and in addition to a ten-fold dilution of the filtrate prepared at a loading rate of 1.0 mg/l for a period of 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken from all test solutions at the start, after 24 hours of exposure and at the end of the test.

Since not all components of Hatcol 5127 were quantifiable in the calibration chromatograms, analytical results were based on the largest peak. Based on this peak the filtered solution prepared at a loading rate of 100 mg/l contained a mean initial concentration of 0.015 mg/l. After 24 and 72 hours of exposure this test concentration had decreased to below the lowest calibration solution ( <0.001 mg/l). The lower test concentrations were below the lowest calibration solution from the start of exposure. Therefore, the toxicological evaluation was based on loading rates.

The study met the acceptability criteria prescribed by the protoco1 and was considered valid.

Due to the fact that test concentrations below or approximating the water solubility level of Hatcol 5127 could not be analysed as these were below the limit of quantification, a different approach was followed for the calculation EC and NOEC-values.

To this respect the following text quoted from the OECD guidance on classification of chemicals is applicable: 'Many substances covered by the classification scheme are in fact mixtures, for which measurement of exposure concentrations is difficult, and in some cases impossible.

Substances such as petroleum distillate fractions, polymers, substances with significant levels of impurities, etc can pose special problems since the toxic concentration is difficult to define and impossible to verify. Typical testing procedures often rely on the formation of a Water Soluble Fraction (WSF) or Water Accommodated Fraction (WAF) and data are reported in terms of loading rates. These data may be used in applying the classification criteria.' (Organization  For Economic Co-operation ad Development (OECD), OECD guidance document on the use of the harmonised system for the classification of chemicals which are hazardous for the aquatic environment, OECD series on testing and assessment number 27, Ju/y 23, 2001).

Hatcol 5127 reduced growth rate of Selenastrum capricornutum significantly at a loading rate of 100 mg/l (both statistical and biological).

 

The table below gives the effect parameters based on the loading rates. Effect parameters based on the initial concentrations are given in the fourth column of this Table.

Parameter	Loading rate Hatcol 5127 (mg/L)	95% confidence interval	Initial conc. Hatcol 5127 (mg/L)
NOELR	10
72hr-EBL10	11	4.7-27
72hr-EBL50	>100	50-300	>0.015
72hr-ERL10	26	15-47
72hr-ERL50	>100	1500-14000	>0.015

Description of key information

Due to the fact that test concentrations below or approximating the water solubility level of Hatcot 5127 could not be analysed as these were below the limit of  quantification, a different approach was followed for the calculation of EC and NOEC-values.

To this respect the following text was quoted from the OECD guidance  on classification of chemicals is  applicable: ‘Many substances covered by the classification scheme are in fact mixtures, for which measurement of exposure concentration is difficult, and in some cases impossible.  Substances such as  petroleum distillate fractions, polymer, substances with significant levels of impurities, etc can pose special problems since the toxic concentration is difficult to define and impossible to verify. Typical testing procedures often rely on the formation of a Water Soluble Fraction (WSF) or Water Accommodated Fraction (WAF) and data are reported in terms of loading rates. These data may be used in applying the classification criteria. Organisation for Economic Co-operation and Development (OECD), OECD guidance document on the use of the harmonised system for the classification of chemicals which are hazardous for the aquatic environment, OECD series on testing and assessment number 27, July 23 2001)

Under the conditions of the present study with Selenastrum capricornutum, Hatcol 5127  reduced growth rate of this fresh water algae species significantly at a loading rate of 100 mg/l (both statistical and biological).

The EL50 for cell growth inhibition (EBL50: 0-72h) was beyond the range tested.

The EL10 for cell growth inhibition (EBL10: 0-72h) was 11 mg/l with a 95 % confidence interval  ranging from 4.7 to 27 mg/l.

As growth rate is derived from the slope under the growth curve in a logarithmic plot, the  measure of the specific growth rate is preferable over biomass following from the mathematical nature of exponential growth.

The EL50 for growth rate reduction (ERL50: 0-72h) was beyond the range tested.

The EL10 for growth rate reduction (ERL10: 0-72h) was 26 mg/l with a 95 % confidence interval

ranging from 15 to 47 mg/l.

The NOELR for both cell growth inhibition and growth rate reduction was 10 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

 

Selenastrum capricornutum, fresh water algal growth inhibition test with Hatcol 5127.

The study procedures described in this report were based on the ISO international standard 8692 1989. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.3, 1992, the OECD guideline No 201 1984 and the OECD series on testing and assessment number 23 December 14 2000.

Hatcol 5127 is a light yellow liquid. The water solubility of Hatcol 5127 at 20.4 +/- 0.7˚C was determined to be < 0.14 x 10-3 g/l in a preliminary experiment (pH was between 8.0-8.1, NOTOX Project 388035).

 

All test solutions with a loading rate at or above 1.0 mg/l were prepared separately (loading rates of 1.0, 10 and 100 mg/l). These saturated solutions were stirred for three days to reach maximum solubility. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance (see also NOTOX Project 388035). After the stiring period all three mixtures/solutions were therefore filtered twice. First through a glass fiber pre-filter to remove the larger undissolved test substance particles and subsequently through a membrane filter (0.45µm). Finally a ten-fold dilution was prepared from the filtrate prepared at a loading rate of 1.0 mg/l. All test solutions were clear and colourless.

A final test was performed exposing exponentially growing algae to 0.45µm filtered solutions prepared at loading rates of 1.0, 10 and 100 mg/l and in addition to a ten-fold dilution of the filtrate prepared at a loading rate of 1.0 mg/l for a period of 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken from all test solutions at the start, after 24 hours of exposure and at the end of the test.

Since not all components of Hatcol 5127 were quantifiable in the calibration chromatograms, analytical results were based on the largest peak. Based on this peak the filtered solution prepared at a loading rate of 100 mg/l contained a mean initial concentration of 0.015 mg/l. After 24 and 72 hours of exposure this test concentration had decreased to below the lowest calibration solution ( <0.001 mg/l). The lower test concentrations were below the lowest calibration solution from the start of exposure. Therefore, the toxicological evaluation was based on loading rates.

The study met the acceptability criteria prescribed by the protoco1 and was considered valid.

Due to the fact that test concentrations below or approximating the water solubility level of Hatcol 5127 could not be analysed as these were below the limit of quantification, a different approach was followed for the calculation EC and NOEC-values.

To this respect the following text quoted from the OECD guidance on classification of chemicals is applicable: 'Many substances covered by the classification scheme are in fact mixtures, for which measurement of exposure concentrations is difficult, and in some cases impossible.

Substances such as petroleum distillate fractions, polymers, substances with significant levels of impurities, etc can pose special problems since the toxic concentration is difficult to define and impossible to verify. Typical testing procedures often rely on the formation of a Water Soluble Fraction (WSF) or Water Accommodated Fraction (WAF) and data are reported in terms of loading rates. These data may be used in applying the classification criteria.' (Organization  For Economic Co-operation ad Development (OECD), OECD guidance document on the use of the harmonised system for the classification of chemicals which are hazardous for the aquatic environment, OECD series on testing and assessment number 27, Ju/y 23, 2001).

Hatcol 5127 reduced growth rate of Selenastrum capricornutum significantly at a loading rate of 100 mg/l (both statistical and biological).

 

The table below gives the effect parameters based on the loading rates. Effect parameters based on the initial concentrations are given in the fourth column of this Table.

Parameter	Loading rate Hatcol 5127 (mg/L)	95% confidence interval	Initial conc. Hatcol 5127 (mg/L)
NOELR	10
72hr-EBL10	11	4.7-27
72hr-EBL50	>100	50-300	>0.015
72hr-ERL10	26	15-47
72hr-ERL50	>100	1500-14000	>0.015