AFLATOXIN CONTAMINATION AND MOISTURE LEVELS IN SRI LANKAN

r Samples of raw and parboiled, milled rice collected from wet (Veyangoda), intermediate (Kumnegda) and dry (Anuradhapura) zones of Sri Lanka during the dry and rainy seasons of the year were analysed for the presence of moisture and aflatoxin B1. Rice contained a mean moisture content of 14.9 f 1.4%. Although slightly higher average moisture levels were observed during the rainy season and in the wet zone, the differences in the moisture levels between the two seasons and among the three locations were not statistically significant. Of the 597 samples of rice tested, aflatoxin B1 was not detected in 525 samples (minimum detectable level 12 /&/kg). Aflatoxin B1 was present at 30 W g level in 12 parboiled samples and 60 samples contained 12-30 /&/kg. Of the 60 samples 57 were parboiled rice. At present, rice does not appear to pose a danger through aflatoxin contamination as it is stored only for about a week at moisture levels around 15%. However, with self sufficiency in rice resulting in longer durations of storage, the parboiled rice could indicate a potential danger, if efficient drying methods are not practised.


Introduction toxins are the most potent naturally occurring hepatocarcinogens in experimental anirnalse6
The aflatoxin producing fungi Aspergillus flavus and Aspe?''llus parasiticus have been reported t o grow and produce aflatoxins on many agricultural food commodities, specially under tropical climatic conditidfis and in the presence of moisture levels above 1 0 per cent.Under experimental conditions rice has been shown to be a good medium for production of aflatoxin~.~In rice, moisture levels a b o v ~l 0 per cent c-o@d__q_ccur due-to underdrying of paddy (rough rice), especiaiiy during tne prvcessing of paruuueu rice and also due to absorption of moisture from the atmosphere at high relative humidities during storage.
The growth of fungi in market rice is a common phenomenon and the presence of aflatoxins in rice has been observed in several South-East Asian c o ~n t r i e s .~ There are reports of several incidents of human deaths suspected to be due to aflatoxicosis caused by the consumption of aflatoxin contaminated rice.3 Rice is the staple diet of Sri Lankans.The contamination of rice by aflatoxins even at low levels could be critical as considerable quantities of rice are consumed by the average Sri Lankan.
In view of the relevance of the rice-aflatoxin problem to human health a survey of aflatoxin contamination in rice was carried out in three locations representing the three different climatic zones in Sri Lanka during dry and wet seasons of the year, t o assess the extent of contamination andrelate it t o possible factors which could support fungal contamination and aflatoxin accumulation.In this survey, only aflatoxin B1, which is the most toxic and most widely spread in foods, was estimated.

Source of samples
Samples of all available types of rice, consisting of parboiled and raw milled rice of locally grown varieties were collected from more than 90 per cent of the market places in the three towns Anuradhapura (dry zone, annual rainfall < 2000 mm) Kurunegala (intermediate zone, annual rainfall 2000 -2500 mm) and Veyangoda (wet zone, annual rain fall > 2500 mm).
Samples were collected during the dry and rainy seasons of the year.

Sampling
When sampling was done from 1-5 bags, each containing upto 65 kg, several primary samples were withdrawn from three different levels in the bag using a probe grain sampler.A composite sample was prepared by pooling the three primary samples.A replicate fraction (125 g) from one-fourth of the composite sample was used for laboratory analysis.When the number of rice bags available for sampling was between 6-25,5 bags were picked randomly and primary samples were withdrawn from them.When the number of bags was over 25, they were marked into lots of 5 bags each and a sample was obtained from one bag in each lot.
Both parboiled and raw rice were sampled for analysis.The samples were packed and sealed in polythene bags for transport and storage.

Estimation of moisture
The moisture content in the rice (wet weight basis) was estimated immediately on return t o the laboratories (within 24 hours of collection) using a Kett PBTK moisture meter.The grain samples in sealed polythene bags were stored-at -20°c pending assay for aflatoxins.

Estimation of aflatoxins
Rice grains were ground to a fine powder in a Stein Mill Model M2.The samples in duplicate (20 g) were extracted by the modified 70% aqueous acetone procedure.4Theestimation of aflatoxin B1 was done on 250 nm ' silica gel G ( ~e r c k ) thin layer chromatographic plates by visual comparison with standard samples of pure aflatoxin B1 (Makor, Jerusalem) in chloroform under UV light at 365 nm.The minimum detectable level under these conditions was 12 ~lglkg (ppb).

Locations
In the selection of localities for the survey, three rice producing areas were identified for sampling, as our preliminary studies indicated that it was not possible to find the area of origin of the rice, with reasonable accuracy, if sampling was done in non-rice producing areas of the country.In the three areas surveyed, all rice samples were found t o be produced and processed in the same locality.In 99 per cent of the samples, the storage period as market rice was found to be less than 7 days.Occasionally, samples stored for 14-21 days were present.
hi2 Moisture levels The mean moisture levels observed for all the samples was 14.9 + 1.4%.Lowest moisture levels were observed in rice during the dry season in the intermediate zone (Kurunegala) and highest during the rainy season in the dry zone (Anuradhapura) (Table 1).
In the dry and intermediate zones, the mean moisture content in rice appeared to be higher during the rainy season than during the dry season, whereas no differences in the mean moisture content was observed during the dry and rainy seasons in the wet zone.
The distribution pattern of moisture levels in rice (Table 2) showed an increase in the number of samples containing higher moisture levels during the rainy season in all three localities.The differences were marked in the dry zone.
However, the analysis of variance (Table 1) did not show any significant differences between moisture levels during cQy and rainy seasons and among the three locations.On the whole the average moisture content appeared to be sufficient for slow growth of fungi in rice.

Aflatoxin content
Of 597 samples of rice tested for the presence of aflatoxins none of the samples contained aflatoxin B1 above the UNICEF/WHO/FAO maximum permissible level of 30 pg/kg (ppb) for foods for human consumption.
Aflatoxin B1 was not detected in 525 samples (minimum detectable level , 12 pg/kg).Of the balance there were 12 p@rh?iledrice samples containing 30 pg/kg -of aflatoxii B1.Among the 60 samples which c o n t e e d less than Parboiled rice is more likely to be colonized by the fmgi due to high moisture levels in them caused by improper drying, after parboiling.The gelatinized starch of the parboiled rice endosperm may also be more susceptible to fungal attack than the native starch of the raw endosperm.
As regards the susceptibility of raw rice, the number of samples analysed is not sufficient for a final conclusion.It is therefore not possible to conclude whether the parboiled rice is more susceptible t o aflatoxin accumulation.
The aflatoxin B1 levels observed in the rice samples did not show any pattern of association with the different agro-climatic zones or the seasons indicating that the observed mean moisture levels up to 16% do not influence aflatoxin accumulation in rice during storage.
Under the present conditions where rice is not stored for periods above 7 days at moisture content above 16%, rice doesnot appear to pose a danger through aflatoxin contamination.However, the low aflatoxin levels detected in parboiled rice could indicate a potential danger specially if efficient drying is not practised after parboiling and prior t o storage.
At present rice is a fast selling commodity in the market as Sri Lanka has not reached self sufficiency in rice.However, with self sufficiency in rice, which is expected in the near future, we may face problems of aflatoxin accumulation associated with longer periods of storage and at higher moisture levels as observed with prolonged storage of improperly dried rough riceal