Reaction product of ammonium molybdate and C12-C24-diethoxylated alkylamine (1:5-1:3)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 November 2002 to 11 November 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of ammonium molybdate and C12-C24-diethoxylated alkylamine (1:5-1:3)

EC Number:

412-780-3

EC Name:

Reaction product of ammonium molybdate and C12-C24-diethoxylated alkylamine (1:5-1:3)

Cas Number:

1380226-46-9

Molecular formula:

A complex mixture of species so no unique molecular formula can be given

IUPAC Name:

dimolybdenum(6+) diammonium 2-{2-[(octadec-9-en-1-yl)amino]ethoxy}ethan-1-ol heptaoxidandiide

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): ADDITIV 104
- Physical state: dark red liquid
- Lot/batch No.: 651010903
- Storage condition of test material: room temperature, in the dark

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not reported

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours.

Test solutions

Vehicle:

no

Details on test solutions:

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

Using syringes amounts of test material (125, 250, 500, 1000 and 2000 mg) were each separately added onto the surface of 10 litres of culture medium to give the 12.5, 25, 50, 100 and 200 mg/l loading rates respectively. These were then stirred for 23 hours using a stirring rate such that a
dimple was formed at the media surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A small amount of each WAF was then removed and checked for the presence of micro-dispersions or globules of test material. As globules/micro-dispersions of test
material were observed to be present it was considered appropriate to remove the aqueous phase or WAF by filtration through a glass wool plug.

A glass tube, covered at one end with paraflim was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom ofthe vessel. A length of Tygon tubing was then inserted into the glass tube and pushed through the parafilm seal. At the opposite end of the
Tygon tubing a glass wool plug was inserted (approximately 2-4 cm in length) to trap any micro-dispersions. The aqueous phase or WAF was then removed by filtration through the glass wool plug (the first 75-100 ml discarded) and used for testing.

Microscopic examination of the WAFs after filtration through a glass wool plug showed there to be no globules/micro-dispersions of test material present.

An aliquot (700 ml) of each of the WAFs was separately inoculated with algal suspension (3.5 ml) to give the required test concentrations of 12.5, 25, 50, 100 and 200 mg/l loading rate WAF.

The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72

Post exposure observation period:

None

Test conditions

Hardness:

Not reported

Test temperature:

Temperature was maintained at 24 ± 1°C throughout the test.

pH:

Test start: 7.7
Test end: 8.5
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter

Dissolved oxygen:

Not reported

Salinity:

Not reported

Nominal and measured concentrations:

Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 12.5, 25, 50, 100 and 200 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks plugged with polyurethane foam bungs to reduce evaporation.
- Aeration: constant
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.06 x 106 cells per ml. Inoculation of 700 ml of test medium with 3.5 ml of this algal suspension gave an initial cell density of 10 cells per ml and had no significant dilution effect.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

CULTURE MEDIUM
NaNO3 25.5 mg/l
MgC12.6H20 12.164 mg/l
CaCI2.2H20 4.41 mg/l
MgSO4.7H20 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/I
H3B03 0.1855 mg/l
MnCi2.4H20 0.415 mg/l
ZnC12 0.00327 mg/l
FeCi3.6H20 0.159 mg/l
CoC12.6H20 0.00 143 mg/l
Na2MoO4.2H20 0.00726 mg/l
CuC12.2H20 0.000012 mg/l
Na2EDTA.2H20 0.30 mg/l
Na2SeO3.5H20 0.000010 mg/I

The cuiture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl. The prepared media was sterilised by 0.2 micron membrane flitration

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality:approximately 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter®
Multisizer II Particle Counter.
- Range finding study: Yes
- Results used to determine conditions for the definitive test: Yes

TEST CONCENTRATIONS
- Range finding study
- Test concentrations (Initial range finding test): Water Accommodated Fractions (WAFs) of the test material, nominal loading rates of 100 and 1000 mg/l
- Test concentrations (Second range finding test): Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 1.0, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated over a 72-Hour period and gave an EbL*50 (72 h) value of 26 mg/l loading rate WAF; 95% confidence limits 22 -31 mg/l loading rate WAF and an ErL*50 (0 - 72 h) value of 56 mg/l loading rate WAF;95% confidence limits 50-62 mg/l loading rate WAF.

The No Observed Effect Loading rate at 72 hours was 12.5 mg/l loading rate WAF

Any other information on results incl. tables

Inhibition of Growth Rate and Biomass

Nominal Loading Rate (mg/l)  Area Under Curve at 72h  % Inhibition  Growth Rate 0 -72 h)  % Inhibition Control   2.81E+07 -   0.071  -  12.5  2.90E+07  [3]  0.073  [3]  25  1.52E+07  46  0.064  10  50  4.44E+06  84  0.045  37  100  2.98E+04  100  0.000  100  200  -8.20E+04  100  -0.008  111

[Increase in growth as compared to the controls] E_bL₅₀(72 h) from Figure 2 = 26 mg/l loading rate WAF

E_rl₅₀(0 - 72 h) from Figure 2 =56 mg/1 loading rate WAF

Applicant's summary and conclusion

Conclusions:

The No Observed Effect Loading rate (NOELR) at 72 hours was calculated to be 12.5 mg/l loading rate WAF.

Executive summary:

In a GLP compliant, guideline 72 hour acute toxicity test to aquatic algal species Scenedesmus subspicatus the test substance gave an EbL*50 (72 h) value of 26 mg/l loading rate WAF; 95% confidence limits 22 -31 mg/l loading rate WAF and an ErL*50 (0 - 72 h) value of 56 mg/l loading rate WAF; 95% confidence limits 50 -62 mg/l loading rate WAF.

The No Observed Effect Loading rate at 72 hours was 12.5 mg/l loading rate WAF.