Paraffin waxes and Hydrocarbon waxes, oxidized, lithium salts

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:

10th May 2012 to 15th June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom.

Analytical monitoring:

yes

Details on sampling:

-Samples of the uninoculated control and 100 mg/l loading rate WAF (Water Accommodated Fraction) test groups were taken for analysis at 0 and 72 hours.
-Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing and following a 1-Hour settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

RANGE FINDING TEST
Amounts of test item (20 and 200 mg) were each separately weighed onto a glass slide and suspended within 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 - 100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

DEFINITIVE TEST
An amount of test item (200 mg) was weighed onto a glass slide and suspended within 2 litres of culture medium to give the 100 mg/l loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 - 100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Liquid cultures were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 °C.

ACCLIMATION
- Culturing media and conditions (same as test or not): Yes. Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/ml.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

7.7 - 8.0

Salinity:

Not reported

Nominal and measured concentrations:

Nominal loading rate 100 mg/l

Details on test conditions:

CULTURE MEDIUM
-The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

PROCEDURE

Range-finding test
-Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a WAF.
-The loading rate to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.
-The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.

-An aliquot (500 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (11.3 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.
-The control group was maintained under identical conditions but not exposed to the test item.

-At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
-After 72 hours the cell density of each flask was determined using a Coulter Multisizer Particle Counter.


DEFINITIVE TEST

Experimental Preparation
-An aliquot (1 litre) of the WAF was inoculated with algal suspension (19.5 ml) to give the required test concentration of 100 mg/l loading rate WAF.

Exposure conditions
-As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and 100 mg/l loading rate WAF treatment group.
-The control group was maintained under identical conditions but not exposed to the test item.

-Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.56 x 10^5 cells per ml. Inoculation of 1 litre of test medium with 19.5 ml of this algal suspension gave an initial nominal cell density of 5 x 10^3 cells per ml and had no significant dilution effect on the final test concentration.
-The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
-Samples were taken at 0, 23, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter.


Vortex depth measurements
-The vortex depth was recorded at the start and end of the mixing period.


EVALUATION OF DATA
-Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:
µ = (lnNn - lnN1) / tn - t1
Where:
µ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.
In addition, the section by section specific growth rate (days 0 - 1, 1 - 2 and 2 - 3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.
Percentage inhibition of growth rate for each replicate test item vessel was calculated using the following equation:
Ir = [(μc - μt) / µc] x 100
Where:
Ir = percentage inhibition of average specific growth rate
µc = mean average specific growth rate for the control cultures
µt = average specific growth rate for the test culture

- Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:
Y = Nn - N0
Where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:
Iy = [(Yc - Yt) / Yc] x 100
Where:
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group

-Comparison of Biomass Integral
The biomass integral (area under the growth curve) was calculated using the following equation:
A = [(N1 - N0) / 2] x [t1 + ((N1 + N2 - 2N0) / 2)] x [(t2 - t1) + ((Nn-1 +Nn - 2N0) / 2)] x (tn - tn-1)
Where:
A = area
N0 = nominal cell concentration at start of test
N1 = measured cell concentration at t1
Nn = measured cell concentration at tn
t1 = time of first measurement after beginning of test
tn = time of nth measurement after beginning of test.

Percentage inhibition of the biomass integral for each replicate test material vessel was calculated using the following equation:
Ia = [(Ac - At) / Ac] x 100
Where:
Ia = percentage inhibition of the biomass integral
Ac = mean biomass integral for the control cultures
At = biomass integral for the test culture

-Determination of ELx Values
Effective Loading Rate values were determined by inspection of the growth rate, yield and biomass integral data after 72 hours.

- Statistical Analysis
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and the 100 mg/l loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
-The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
-The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0 - 1, 1 - 2 and 2 - 3, for 72-hour tests) must not exceed 35 %.
-The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7 %.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

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: WAF

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WAF

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: WAF

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: WAF

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: WAF

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: WAF

Details on results:

RANGE-FINDING TEST
-The results showed no effect on growth at 10 and 100 mg/l loading rate WAF.

DEFINITIVE TEST
-Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 1. Growth rate, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 2.
-From the data given in Tables 1 and 2, it is clear that the growth rate (r), yield (y) and biomass integral (b) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test item over the 72-Hour exposure period.
-It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Observations on cultures
-All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Observations on test item solubility
-Observations on the test media were carried out during the mixing and testing of the WAF. At both the start and end of the mixing period, and following a 1-Hour standing period, the 100 mg/l loading rate WAF was observed to have formed a clear colourless media column with test item adhered to the glass slide suspended within the media column. Microscopic examination of the WAF showed there to be no globules or micro-dispersions of test item present.
-At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.

Total organic carbon analysis
-Total Organic Carbon (TOC) analysis of the 100 mg/l loading rate WAF test preparation at 0 hours showed a measured concentration of 1.64 mg C/l was obtained whilst at 72 hours a measured test concentration of less than the limit of quantitation (LOQ), which was considered to be 1.0 mg C/l was obtained.
-Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole the results were based on nominal loading rates only.

Validation criteria
-The following data show that the cell concentration of the control cultures increased by a factor of 60 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Mean cell density of control at 0 hours: 5.23 x 10^3 cells per ml
Mean cell density of control at 72 hours: 3.16 x 10^5 cells per ml
-The mean coefficient of variation for section by section specific growth rate for the control cultures was 34 % and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35 %.
-The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 5 % and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Results with reference substance (positive control):

-A positive control used potassium dichromate as the reference item at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
-Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
-Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the following results:
ErC50 (0 – 72 h): 0.79 mg/l*
EyC50 (0 – 72 h): 0.49 mg/l, 95 % confidence limits 0.48 – 0.50 mg/l
* It was not possible to calculate 95 % confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

NOEC based on growth rate: 0.25 mg/l
NOEC based on yield: 0.50 mg/l
LOEC based on growth rate: 0.50 mg/l
LOEC based on yield: 1.0 mg/l

-The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

Statistical analysis of the growth rate, yield and biomass data was carried out for the control and 100 mg/l loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There was no statistically significant decrease in growth rate, yield or biomass integral (P≥0.05), between the control and 100 mg/l loading rate WAF test group and therefore the NOELR was 100 mg/l loading rate WAF.

Table 1 Cell Densities and pH Values in the Definitive Test

Nominal Loading Rate (mg/l)		pH	Cell Densities* (Cells per ml)				pH
		0 h	0 h	23 h	48 h	72 h	72 h
Control	R1 R2 R3 R4 R5 R6 Mean	8.0	5.68 E+03 5.09 E+03 5.15 E+03 5.18 E+03 5.07 E+03 5.20 E+03 5.23 E+03	3.90 E+04 3.31 E+04 3.09 E+04 2.98 E+04 2.61 E+04 2.82 E+04 3.12 E+04	1.02 E+05 1.15 E+05 1.09 E+05 1.06 E+05 1.07 E+05 1.09 E+05 1.08 E+05	3.18 E+05 3.15 E+05 2.51 E+05 2.56 E+05 4.81 E+05 2.75 E+05 3.16 E+05	7.9
100	R1 R2 R3 R4 R5 R6 Mean	7.7	5.78 E+03 5.42 E+03 4.66 E+03 5.06 E+03 4.93 E+03 5.22 E+03 5.18 E+03	4.27 E+04 3.78 E+04 4.38 E+04 3.95 E+04 3.64 E+04 3.42 E+04 3.91 E+04	1.20 E+05 1.16 E+05 1.29 E+05 1.25 E+05 9.90 E+04 1.06 E+05 1.16 E+05	5.99 E+05 4.52 E+05 5.07 E+05 5.81 E+05 4.77 E+05 4.49 E+05 5.11 E+05	7.8

*Cell densities represent the mean number of cells per ml, calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1 - R6 = replicates 1 to 6

 

Table 2 Inhibition of Growth Rate, Yield and Biomass Integral in the Definitive Test

Nominal Loading Rate (mg/l)		Growth rate (cells/ml/hour)		Yield (cells/ml)		Biomass Integral (cells/ml/hour)
		0 - 72 h	% Inhibition	0 - 72 h	% Inhibition*	0 - 72 h	% Inhibition
Control	R1 R2 R3 R4 R5 R6 Mean SD	0.058 0.058 0.054 0.055 0.063 0.056 0.057 0.003	-	3.13 E+05 3.10 E+05 2.45 E+05 2.51 E+05 4.76 E+05 2.70 E+05 3.11 E+05 8.58 E+04	-	6.69 E+06 7.09 E+06 6.12 E+06 6.08 E+06 8.71 E+06 6.34 E+06 6.88 E+06 9.91 E+05	-
100	R1 R2 R3 R4 R5 R6 Mean SD	0.066 0.063 0.064 0.066 0.063 0.062 0.064 0.002	[16] [11] [12] [16] [11] [9] [13]	5.93 E+05 4.47 E+05 5.03 E+05 5.76 E+05 4.72 E+05 4.44 E+05 5.06 E+05 6.47 E+04	[63]	1.09 E+07 8.88 E+06 1.00 E+07 1.07 E+07 8.72 E+06 8.51 E+06 9.60 E+06 1.03 E+06	[58] [29] [45] [55] [27] [24] [40]

*In accordance with the OECD test guideline, only the man value for the yield is calculated.

R1 - R6 = replicates 1 to 6

SD = Standard Deviation

[Increase in growth as compared to controls]

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this test, the effect of the test material on the growth, yield and biomass of Desmodesmus subspicatus gave EL50 values of greater than 100 mg/l loading rate WAF. In addition, the NOELRs were 100 mg/l loading rate WAF.

Executive summary:

In a GLP compliant study performed according to the standardised guidelines OECD 201 and Method C.3 of Commission Regulation (EC) 761/2009 the toxicity of the test material was determined in an Algal Growth Inhibition Test.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test the test material was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a WAF of the test item at a single nominal loading rate of 100 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Exposure to the test material gave EL50 values of greater than 100 mg/l loading rate WAF. The No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole the results were based on nominal loading rates only.

Description of key information

The toxicity of the test material was determined in Desmodesmus subspicatus. Under the conditions of the test, no toxic effects were noted and the 72-Hour EL50 based on nominal loading rates was greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The potential for the test material to cause acute toxic effects to aquatic plants was determined in a GLP compliant study (Vryenhoef 2012) performed according to the standardised guidelines OECD 201 and Method C.3 of the Commission Regulation (EC) No. 761/2009. The study was reported in sufficient detail so as to accurately represent the toxicity of the material.

The toxicity of the test material was determined in an Algal Growth Inhibition Test. Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test the test material was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a WAF of the test item at a single nominal loading rate of 100 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Exposure to the test material gave EL50 values of greater than 100 mg/l loading rate WAF. The No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole the results were based on nominal loading rates only.

The study was performed to a good standard and was assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch (1997).